Special Beport Series. No. 121. ^ribg (ffouncil MEDICAL RESEARCH COUNCIL 3 9424 00444 9358 Borna DiSBSSrtHH^ffizootic Encephalo- Myelitis of Sheep and Cattle BY S. NICOLAU, M.D., D.Sc., and I. A. GALLOWAY, B.Sc, M.R.C.V.S. "B.C. LIBRARy SF799 LONDON IS MAJESTY'S STATIONERY OFFICE 1928 Price 5s. net FORAG • Sped U.B.C. Library \ Report Series, No. 121. j CAT HO. P^'nX:.,!±i J ^ ACC. NO. '}i 9^^^— J MEDICAL RES ^^^^^^f^f^—-- — COUNCIL Borna Disease and Enzootic Encephalo- Myelitis of Sheep and Cattle BY S. NICOLAU, M.D., D.Sc, and I. A. GALLOWAY, B.Sc, M.R.C.V.S. LONDON PUBLISHED BY HIS MAJESTY'S STATIONERY OFFICE 1928 45-5-2I. MEDICAL RESEARCH COUNCIL The Right Hon. Earl of Balfour. K.G.. O.M.. F.R.S. {Chairmnn). The Right Hon. Lord Mildmay of Flete. P.C. (Treasurer). The Right Hon. William Graham, LL.B., M.P. Sir Hugh K. Anderson, M.D., F.R.S. Professor E. P. Cathcart, C.B.E., M.D., D.Sc, F.R.S. Professor G. Dreyer, C'.B.E.. M.D., F.R.S. Professor T. R. Elliott, C.B.E., D.S.O., M.D., F.R.S. Sir Archibald E. Garrod, K.C.M.G., D.M., F.R.S. Sir Frederick G. Hopkins, D.Sc, F.R.S. Sir Charles J. Martin, C.M.G., D.Sc, F.R.S. Sir Charles S. Sherrington, O.M., G.B.E., F.R.S. Sir Walter M. Fletcher, K.B.E., M.D., ScD., F.R.S. {Secretary PREFACE The present report, which was received for publication in October, 1927, gives the results of investigations made by Dr. Xicolau and Mr. Galloway, working as guests in the National Institute of Medical Research. These studies of the virus which is responsible for a danger- ous communicable disease in horses, cattle and sheep have happily no immediate practical apphcation in this country. Borna disease and its congeners are at present only known in continental Europe and in America. Our present immimity, however, may only be temporary, and in any case it is highly important that we should have the fullest knowledge of these epizootic diseases of other countries. The scientific advantages gained by close association between studies of disease in animals and studies of human disease have long been obvious. The potential value to medical science of the work now presented Ues in two directions. The accurate experimental study of the ' virus ' which is the causal agent in this disease is part of the general study of disease viruses, and in this field of work great gain must come from the free exchange of ideas, methods and results among workers in different parts of it. Besides this, however, Borna disease has special points of interest to students of human neurology. The infective virus produces changes in the brain and spinal cord, the so-called encephalo-myehtis, which throw light upon analogous forms of encephahtis and myehtis which occur in sporadic or epidemic form in human beings. It will be seen that the authors have extended or confirmed the observations of many previous workers, and have gained new know- ledge at several points of detail by their experimental work. Fresh studies have been made of the immunity reactions of the virus, and it has been shown that animals may be successfully immunized against it. Dr. Nicolau was enabled to conduct this work in the National Institute by a grant from the Roumanian Government. Mr. Galloway co-operated with him in the course of other work upon foot and mouth disease in cattle, which he is doing on behalf of the Ministry of Agri- culture and Fisheries. The council are indebted to the Ministry for the faciUties they have given for this co-operation. 28th July, 1928. Medical Research Council, 15 York Buildings, London, W.C. 2. 1.) Ps. 28720. Wt. 15076. 536/970. 1250. 9/28. O.U.P. BORNA DISEASE AND ENZOOTIC ENCEPHALO- MYELITIS OF SHEEP AND CATTLE By S. Nicolau. M.D., D.Sc, and I. A. Galloway, B.Sc, M.R.C.V.S. (Xational Ivstilute for Medical Research. Hampstead.) CONTENTS PAOE 1. HiSTORiCAi, AXD General ......... 7 I. Enzootic Encephalo-myelitis of the Horse ...... 7 II. Enzootic Encephalo-myelitis of Cattle ...... 9 III. Enzootic Encephalo-myelitis of Sheep ...... 10 IV. Malignant Catarrhal Fever of Cattle . . . . . .11 V. Summary . . . . . . . . . . .11 2. Pbopekties of the Virus ......... 12 I. In\-isibility ........... 12 II. Filterability: Effect of Dilution 12 III. Centrifugalization .......... 15 IV. Resistance to Glycerine ......... 17 V. Sensitiveness to Heat and Desiccation ...... 20 \^. Action of Utra-violet Light 20 VII. Action of Hexamethylenetetramine (Urotropine) . . . .21 VIII. Action of Chloroform and Ether ....... 21 IX. Action of Formalin ......... 22 X. Culture 22 XI. Summary ........... 22 3. Tkassmission of Eqiixe Strain to Babbit and from Rabbit to Sheep A>'D VICE versa .......... 23 4. ExpERniEXTAL Disease rx the Rabbit ....... 2t I. Experimental Transmission of the Disease to the Rabbit . . .24 II. Authors' Observations ......... 24 III. Symptomatology of the Disease in the Rabbit . . . . .27 IV. Routes by which Rabbits can be Infected . . . . . .29 5. AriHORS' EXPEKDIESTS ON THE TbASSMISSIOS OF THE DISEASE TO MONKEYS, AND SvilPTOMS OCCUKKING IN THESE ANIMALS . . . . .34 6. Pathogenicity of the Virus of Enzootic Encephalo-myelitis fob the Guinea-pig, Rat, Mouse, and Fowl ....... 39 I. Guinea-pig ........... 39 II. Rat 43 III. Mouse 44 IV. Fowl 44 7. Animals which have bees found to be Resistant to Infection with the Virus of Enzootic Encephalo-myelitis ...... 44 I. Dog 44 II. Pigeon ............ 44 III. Ferret 44 8. Distribution OF the Virus of Borsa Disease IN THE Animal Body . . 44 I. Passage of Virus through the Placenta . . . . . .44 II. Distribution of the Virus in Various Organs and Tissues . . .45 III. Presence of Virus in the Peripheral Nerves of Rabbits inoculated Intra- cerebrally .......... 45 IV. Distribution of the Virus in various Organs and Tissues of the Monkey . 48 6 CONTENTS PAGE 9. EUMUJATION OF THE VlRUS FROM THE AsTMAL OrGASISM . . . .49 10. HiSTOPATHOLOGY OF BoRSA DISEASE ....... 50 I. Horse 50 II. Cattle 50 III. Sheep 51 IV. Experimental Borna Disease in Horses and Sheep . . . .51 V. Rabbits infected Experimentally ....... 51 \"I. -Authors' Observations . ........ 52 A. Babbit 52 (1) ilacroscopical and Microscopical Findings in Diverse Organs 52 (2) Lesions in the Central Servoiis System . . . .53 (a) The Brain 53 (b) The Midbrain and Medulla Oblongata . . .58 (c) The Cerebellum 59 (d) The Spinal Cord 59 (3) Lejiions in the Peripheral Xerrous System . . .60 (a) The Posterior Xerie Boots tiO (b) The Spinal Ganglia tiO (c) The Peripheral Xerves . . . . . .1)1 (4) Summary and Discussion . . . . . .62 B. The Guinea-pig 64 C. The Bat and Mouse ........ 64 D. The Monkey (H ( 1 ) Lesions in the Central Xenons System . . . .65 (a) Brain ......... 65 (b) Cerebellum ........ 66 (c) Medulla Oblongata ....... 66 (d) .Spina/ Cord 66 (e) Sciatic Xerre ........ 67 (f) Brachial Xerie 68 (2) Summary 68 11. hl.MlNITY 69 1. Attempts at eonferrinn Immunity ttween the Strain of Kquine and tliat of Ovine Origin ......... 75 V. Experiments on Cross Immunity between the Virus of Kncephnlo- myelitis and other Viruses disease is produced by a, virus similar, if not identical, with that which produces liornu dJBeuBe in horses. ENZOOTIC ENCEPHALO-MYELITIS 11 IV. Malignant Catarrhai, Fever of Cattle. Glamser (1926) and Dobberstein (192.5) found perivascular and parenchymatous infiltration as well as alterations in the ganglion cells of the brain of cattle dead of a disease which they called malig- nant catarrhal fever. These lesions were similar to those described in Borna disease. Ernst and Hahn (1927) also draw attention to the similarity of the lesions in the brain of cattle dead of this disease with those found in encephalo-myehtis of the horse, and in 3 out of 5 cases they observed the intranuclear corpuscles of Joest-Degen in the ganglion cells of the Amnion's horn. Emulsions from the brain of one of these cases inoculated intracerebrally into rabbits produced a disease similar to experimental Borna disease and transmissible from rabbit to rabbit. They concluded that malignant catarrhal fever of cattle is produced by a virus which approaches very closely, if it be not identical with, that producing encephalo-myelitis in horses. We might here mention that Ernst and Hahn (1927) also made an observation indicating that deer are susceptible to Borna disease. A sick deer was killed by a hunter under curious circumstances. The animal allowed the hunter to approach very closely, drew away in fear, and then rushed on him suddenly. The ears were seen to twitch and the animal turned round in a circle until the hunter, probably more frightened than the animal, killed it. The head was brought to Munich. The brain showed the presence of lesions similar to those of Borna disease of the horse, and the intranuclear corpuscles of Joest- Degen were observed. Their attempts at transmission of the disease to laboratory animals had given no results at the time of publication of these observations. V. Summary. From the foregoing resume of the literature of spontaneous encephalo-myelitis in different species, the following conclusions may be drawn : (1) The enzootic encephalo-myelitis of horses and cattle and of sheep appears to be the same disease. The symptomatology and the lesions found in the central nervous system are analogous, and the intranuclear corpuscles of Joest-Degen occur in the large ganglion cells of the Amnion's horn in all three species suffering from the disease in question. (2) From cases of all three diseases a virus has been recovered and shown to be responsible for the disease. (3) From the observations of Ernst and Hahn it would seem not improbable that, if the animals had not in addition to malignant catarrhal fever a concomitant infection with Borna disease, some of the cases described as malignant catarrhal fever of cattle were en- cephalo-myehtis. (4) Deer appear to suffer from a similar disease spontaneously. (5) The transmission of the disease under natural conditions is probably by the respiratory tract or by ingestion. 12 BORNA DISEASE AND 2. PROPERTIES OF THE VIRUS I. Invisibility. The virus in ultravisible. Various methods of staining have been used to discover a parasite, but without success. Methods of im- pregnation with silver have not revealed the presence of parasites in the brain of animals dead of the experimental disease in our hands. The existence of the virus in the brain is, however, associated with the presence of intranuclear corpuscles, first described by Joest and Degen (1909). The interpretation of the nature of these corpuscles which we favour is discussed in the chapter dealing with the histo- pathology of the disease, and we are persuaded that they are of the same nature as the similar ' inclusions ' found in other diseases produced by filterable viruses, such as fowl plague, fowl pox, distemper, and 'Virus III' disease of rabbits. II. Filterability: Effect of Dilution. Filtration of emulsions containing viruses through filters which hold back bacteria generally greatly diminishes the concentration of the virus. There are many reasons for tliis, apart from the size of the virus. If the virus is contained in or on cellular particles these may be retained, or the virus itself may be adsorbed on the filter. In such adsorption the hydrogen-ion concentration and the electrical charge carried by the virus exert a decisive influence. The pressure under which the filtration is carried out is also a factor of importance. Further, the sensitiveness of the tissue into which a filtrate is inoculated may influence the decision whether a virus is filterable or not. When a sensitive reagent is employed, the passage of an extremely small quantity may be detected. For instance, the dose of neurovaccinia required to infect a rabbit if inoculated intracerebrally is 1/lOOth to l/lOOOth of that required to produce pustules on apphca- tion to the scarified skin. An experimenter using the former method might conclude that the virus passed a i)acteria-proof filter, one using the latter that it did not. The sensitiveness of tlie tissue into which a filtrate is inoculated is therefore a factor of capital importance often neglected in interpretations as to the filterability or noii-tilti-rability of the virus. The experiments of Moussu (1926), Zwick, Seifried, and Witte (1920), and Ernst and Hahn (1927) show that the virus of IJorna disease can pass through ordinary bacteriological filters, but tliat filtration of the virus is not easily effected. Zwick carried out more than thirty filtration experiments with different filters and inoculati»d more than 100 rabliits with the various tiltrates i)efore he succeeded in infecting tlie animals with a filtrate. We liave carried out two filtration ex])eriments, using the follow- ing technique: from the brain of Rabbit 275, which died on the 82n(l day after cerei>ral inoculation and exiiibited characteristic lesions throughout the nervous system, an eniidsion 1 : :{(• was made with ENZOOTIC ENCEPHALO-MYELITIS 13 physiological saline. The emulsion was placed in the ice-chest for three hours to allow the coarser particles to deposit. The supernatant fluid was filtered by aspiration under low pressure through a Handler filter. Experiment 1. The filtrate was inoculated intracerebrally into Babbits 218a, 168a, 228a, and 201a, on the 15.3.27. During a period of 174 days following the inoculation Eabbit 218a did not show any symptoms, and its weight increased in an even curve by 730 gms. After this period had elapsed the resistance of the animal was tested with fresh passage virus by intracerebral inocu- lation. It died in 37 days after showing typical symptoms of the disease. On microscopic examination of sections of the central nervous system characteristic lesions were found. Rabbit 168a, weighing 700 gms.. and Rabbit 228a, weighing 2,080 gms., also remained well for 174 days following the inoculation, and their weights increased to 1,330 gms. and 2,640 gms. respectively. They were both reinoculated intracerebrally with fresh passage virus on September 5, 1927 (174th day since inoculation), and proved not to be refractory to infection: Eabbit 168a dying 14 days and Eabbit 228a 37 days after inoculation with the test dose. The control rabbit, 25a, inoculated intracerebrally on the same date as the others, died 48 days after infection, and typical lesions were found in the central nervous system, Rabbit 201a was inoculated intracerebrally with filtrate on the 15.3.27. Observations. Dale. Weight in gms. Clinical observations. 15.3.27 1,500 20.3.27 1,620 Normal. 25.3.27 1,660 Normal. 31.3.27 1,.520 Animal appeared to be ill. Rabbit found 2.4.27 dead 18 days after inoculation. On the autopsy no abnormal condition of the organs was observed and the cultures of the brain remained sterile. On microscopic examina- tion slight infiltration and perivascular lesions were found in the brain, mid-brain, and spinal cord, and, in addition, a marked pathological ' satellitism ' of the nerve-cells. Passage was made with the brain of this rabbit to a healthy rabbit. No. 260a, with the results given in detail below: Rabbit 260a, weighing 1,740 gms., was inoculated intracerebrally with an emulsion of the brain of Rabbit 201a. It developed a disease of a recurrent nature and succumbed after the second crisis, 161 days after inoculation. Autopsy. All organs macroscopically normal. Cultures in broth from the brain remained sterile. Microscopic examination. Intense and characteristic lesions were found in the brain and in the cord. The intranuclear corpuscles of Joest-Degen were also found. 14 BORNA DISEASE AND Observations. Daft. Weight in gins. 2.4.27 1,700 19.4.27 1.800 28.4.27 1,900 5.5.27 1,920 12.5.27 1,600 16.5.27 1,570 Clinical observations. Nothing abnormal in animal's condition. Depressed, posterior paresis. Placed on his flank the animal made several vain efforts to recover its normal position. Condition ameliorated — slight paresis of hindquarters. 30.5.27 1.600 Much improved. 13.6.27 1,640 Normal. 22.6.27 1,740 ,, 30.6.27 1,770 15.7.27 1,800 1.8.27 1,700 8.8.27 1,540 Animal ill. 15.8.27 1,300 Paresis of hindquarters. 24.8.27 1,180 „ »* 31.8.27 1,000 ,• 9.9.27 980 Head depressed, paresis t\'pical symptoms of the disease. 12.9.27 820 Placed on the flank, the rabbit made vain efforts at recovering the normal position. 13.9.27 Found dead 161 days after inoculation. Experiment 2. A second filtration experiment was carried out with the same technique. The filtrate through a Mandler filter was inoculated into the brain of four rabbits. 150.\. 15*2.a. "iKU. aiul •231.\. Babbit 231a died accidentally 5 days after inoculation. Rabbit 210.\ remained unaffected and gained progressively in weight. The protocols of Rabbits 150.\ and 152.\ are given below. Rabbit 150 a. Rabbit 152a. Weight In Clinical obseria- Weight in Cli in ical observa- Dale. gms. tions. gms. tions. 15.3.27 1.800 2,200 20.3.27 1,880 2.440 Normal. 31.3.27 1,860 2.4400 28.4.27 2.320 2,660 5.5.27 2..520 2,680 12.5.27 2,420 Slight paresis lie- hind. 2,620 21.5.27 2,340 2,680 30.5.27 2„340 Condition im- proved.- 2,620 •• 5.6.27 2,460 Normal. 2,650 ,. 13.6.27 2,M(» 2.790 ,, 29.6.27 2.700 ., 2.700 15.7.27 2,9<) Therefore only Rabbit 1.50.\ showed sliglit transitory .symptoms which ultimately passed away completely. These symptoms may have been due to the inoculation of a small dose of virus. ENZOOTIC ENCEPHALO-MYELITIS 15 In order to determine whether the 3 rabbits, 150a, 152a, and 210a, had developed any degree of immunity as a result of inoculation with the filtrate, 167 days after the first injection they received intra- cerebrally a virulent emulsion of brain at the same time as a normal rabbit, 25a. which served as a control. Babbit 150a died on the 49th day. Rabbit 152a on the 40th day, and Rabbit 210a on the 40th day, and the control rabbit on the 48th day after inoculation of the test dose. Typical lesions were found in all cases on microscopic examina- tion of sections of brain and spinal cord. Conclusions. The results in our few experiments support the con- clusion of Zwick, Seifried, and Witte (1927) that the virus of Borna disease can pass, though with great difficulty, through bacteriological filters which retain ordinary bacteria, and that the concentration of virus in the filtrate is much reduced. Zwick succeeded in obtaining an active filtrate after filtering a virulent emulsion of brain through a Zsigmondy Bachmann collodion membrane, of which the size of the pore was estimated to be 0-75 /u,. Effect of dilution. Few titration experiments have been made. Zwick records that a virulent emulsion of brain is still capable of pro- ducing the disease in a dilution of 1 : 10,000. III. C'entrifugalization. Experiment 1. A homogeneous emulsion of virulent brain was made, and after the larger particles had been allowed to deposit, the supernatant fluid was pipetted and centrifugaHzed for 5 minutes at 5,400 revolutions per minute. The supernatant fluid after centrifugahzation was carefully pipetted off and inoculated intracerebrally into three rabbits weighing between 1,300 and 1,500 gms. These three rabbits fell ill, showed typical symptoms and died, 39, 48, and 90 days respectively after the inoculation. The characteristic lesions of Borna disease were found in the central nervous system of all three. (a) Rabbit 322b. Weight 1,540 gms. (b) Rabbit 321b. Weight 1,300 gms. (c) Rabbit 320b. Weight 1,500 gms. The inoculation was made on 15.3.27. The protocol of the three rabbits is given below: Rabbit 322b. Date. W ight in gms. Clinical observations. 15.3.27 1,540 23.3.27 1,680 Normal. 25.3.27 1,700 31.3.27 1,700 10.4.27 1,620 ^, 15.4.27 1,580 „ 19.4.27 1,420 Paresis of hind quarters. 28.4.27 1,220 Typical symptoms of the disease. 2.5.27 — Found dead 48 days after inoculation. Culture of the bra n. Negative Microscopic exam 'nation. Intense and characteristic lesions in the central nervous system. 16 BOENA DISEASE AND Rabbit 321b. Date. Weight in gms. Clinieal obseri-ations. 15.3.27 1,300 23.3.27 1,280 Normal. 25.5.27 1,380 31.3.27 1,420 10.4.27 1,480 15.4.27 1,600 19.4.27 1,640 28.4.27 2,000 10.5.27 2,100 21.5.27 2,320 30.5.27 2,080 Paresis of hindquart<'rs. 5.6.27 1 ,800 Typical symptoms of disease. 11.6.27 1,580 Coma. Killed 90 days after infection. Cultures of the brain. Negative. Microscopic ejcamination. Intense and characteristic lesions in the central nervous system. Bahhit 320b. Clinical obsenations. Date. Weight in gm 15.3.27 1,500 23.3.27 1,600 25.3.27 1,620 31.3.27 1,440 10.4.27 1,320 15.4.27 1,320 19.4.27 1,140 23.4.27 1,020 Paresis ? Typical symptoms of the disease. Died 39 days after infection. Cultures from the brain. Negative. Microscopic examination. Mild, but characteristic lesions in the central nervous system. Experiment 2. A virulent emulsion of brain was allowed to deposit, and the super- natant fltiid centrifugalized as in the last experiment (5,400 revs, per minute) for 15 minutes. Two rabbits were inoculated intracerebrally with the supernatant fluid. Both these rabbits developed the disease typically with paralysis, and died 28 and 45 days respectively after the inoculation, showing lesions of a characteristic nature in the central nervous system. (a) Rabbit 324b. Weight 1,150 gms. (b) Rabbit 326"b. Weight 1,150 gms. The inoculation was made on 15.3.27. 'Die protocol oj these two rabbits is recorded below. Rabbit 324b. Clinical ol/seri'ations. Normal. DaU. Weight in gms. 15.3.27 l.l.W 2(1.3.27 1..360 25..3.27 1.400 31. .3.27 1,330 11.4.27 1.140 1.5.4.27 l.ltM) 19.4.27 I,l(i0 28.4.27 1.080 29.4.27 — Commencement of symptoms. Paralysis of hindquarters. Found dead 45 clays after in(H'ulation. Cultures from the brain. Negative. Microscopic examination. The brain and spinal cord showed the presence of typical lesions. ENZOOTIC ENCEPHALO-MYELITIS 17 Rabbit 326b. Date. W eight in gms. Clinical observations. 15.3.27 1,150 20.3.27 1,360 25.3.27 1,440 31.3.27 1,380 11.4.27 1,020 Paresi.s of hindquarters. 12.4.27 — Found dead 28 day.s after inoculation. Microscopic examination. The brain and -spinal cord showed the presence of typical lesions. Conclusion. Centrifugalization for even 15 minutes at 5,400 rev. per minute does not deprive the supernatant fluid of virulence. This fact, in addition to the properties of filterabihty and invisibility of the pathogenic agent suggests that the size of the infective element is excessively small. It is affected by centrifugalization in the same way as other filterable viruses such as those of foot-and-mouth disease, rabies, herpes, vaccinia, and pohomyeHtis. IV. Eesistance to Glycerine. Moussu (1926) found that a portion of brain preserved its virulence at room temperature (July-August, Alfort) for 18 days, but that its pathogenic action was lost after 32 days. According to Zwick the brain of a rabbit preserved its virulence in glycerine for from 4 to 5 months, and in our experiments glycerinated virus kept in the cold room at 4° C. was still virulent after 113, 135, and in one case 161 To find the best conditions for keeping the virus in the cold room the following solutions were tried. (1) Pure glycerine, (2) pure gly- cerine covered with a layer of sterile paraffin oil, (3) glycerine mixed with an equal part of sterile physiological saline, (4) glycerine mixed with an equal part of phosphate saline M/25, pH. 7.6. The brain of Rabbit 77a (dead of Borna disease 48 days after inoculation intra- cerebrally with typical lesions in the C.N.S.) was taken aseptically and divided into four equal portions, and one of the portions placed in each of the four media referred to above. At the end of a certain time a fragment of each portion was taken and an emulsion of it inoculated intracerebrally into rabbits to test its virulence. A table of results is given on page 18. The results recorded in the table on p. 18 show that the virus may remain virulent in the cold room at 4° C. at least 113 days in a medium consisting of pure glycerine, glycerine diluted to 50 per cent, with physiological sahne, or glycerine diluted to 50 per cent, with phosphate saline M/25, pH. 7.6. In two further experiments under similar conditions the virus preserved in glycerine remained virulent for 135 days and 161 days respectively. (1) An emulsion of the brain of Rabbit 25 (dead of Borna disease 35 days after inoculation) which had been kept in glycerine in the ice-chest for 135 days was inoculated intracerebrally into Rabbit 145a. 18 BORXA DISEASE AND e B ' J. . . . . . SO e >, s *i J5 .£ jS ^ J- -■3 Q X .*^ CD C •2 o lO » — O -" CJ m .* ■*»J «■* IN n •^, CO >» "2 S -3 - " : : ' - 2J „^ ^ ■M O 30 ■* la 0 a"« Q si 1 11^ 4- "25 ■S5 55 So M » n » S 0 o (M« «N M « s O O O O o o 00 }l •« IN O X °° S . >"£ cil l-o ■3) «g ■g ^ E?i 3 ii 1.^ ^s cx ■5, "3 "■ 0 .5 c • r — f c 2 .s.s S. g-1 l-s. ^1 j< ■"« « a9 3 .= § W .§■§- i^ 'w^ "i^ i-* ENZOOTIC ENCEPHALO-MYELITIS 19 Rabbit 14o.\. Weight 2,500 gms. Clinical observations. Date. Weight in gms 13.6.27 2,500 22.6.27 2,500 29.6.27 2,500 7.7.27 2,320 14.7.27 1,900 19.7.27 1.550 20.7.27 — First symptoms of the disease. Typical symptoms of the disease in an advanced stage. Died 47 days after inoculation. Cultures from brain. Negative. Microscopic examination. Brain and cord showed presence of intense lesions. (2) Virus (brain of Rabbit 100 dead of enzootic encephalo-myelitis on the 37th day after inoculation) kept in glycerine 161 days in the chest, was inoculated Lntracerebrally into Rabbit 142\ on the 3.6.27. Rabbit 142a. Weight 2,440 gms. Date. Weight in gms. Clinical observations. 13.6.27 2,500 Normal. 22.6.27 2,620 29.6.27 2,440 7.7.27 2,040 Typical symptoms commencing. 14.7.27 1,850 16.7.27 1,600 Died 43 days after inoculation. Cultures from brain. Negative. Microscopically. Characteristic and intense lesions were present in the central nervous system. We have observed that certain of the rabbits infected lntracere- brally with virus kept in glycerine succumbed to the disease at an earher date than rabbits inoculated with an emulsion of fresh virulent brain as is indicated by the following experiment. (3) Rabbit 275 was inoculated lntracerebrally ^ith a virus kept in pure glycerine for 48 days on 10.2.27. Clinical observations. No abnormal symptoms observed. Date. Weight in gms 10.2.27 2,100 19.2.27 2,150 23.2.27 2,040 27.2.27 1,940 5.3.27 1,700 7.5.27 — 9.3.27 1,520 12.3.27 — 13.3.27 1,260 14.3.27 1,220 Commencing paresis of the hind quarters. Paresis of hindquarters more advanced. Animal remained in comer of the cage hunched up, depressed. The paresis was still present. Intense .salivation. Paralysis of the hind quarters. Died 32 dajs after the inoculation. The microscopic examination of sections of brain and cord of Rabbit 142a revealed the presence of intense and characteristic lesions. The following table, which shows the chain of the series of passages of the \'irus from rabbit to rabbit, of which Rabbit 275 forms a con- necting link, demonstrates the fact that although it weighed more than 2,000 gms. it died at an earher date than the other rabbits of the same series. b2 20 BORNA DISEASE AND Inoculated iniracerehrally Interval between inocula- Xo. of Rabbit. with tion and death. 100 Fresh rinis. 37 days. 77a Fresh virus from Rabbit 100. 48 „ 275 Virus from Rabbit 77a kept in glycerine for 48 days. 32 „ 211 Fresh virus from Rabbit 275. 42 „ 212 „ „ „ *4 „ A similar observation has been made a number of times in the course of our experiments, and tliis is recorded as a typical example. Levaditi, Harvier, and Nicolau record similar findings for the virus of herpes, and this has been confirmed more recently by Perdrau. Y. Sensitiveness to Heat and Desiccation. (a) Heat. Zwick and his collaborators found that cerebral emul- sions heated for 5, 10, 15, 20, and 25 minutes respectively at 50° C. preserved their virulence for the rabbit. In some instances a similar emulsion heated for 30 minutes at 50° C. became avirulent. Heated for 30 minutes at 57° C. or 10 minutes at 70° C. in the water-bath the virulence of the emulsion was destroyed. (b) Desiccation. Zwick found that a virulent emulsion of brain dried for 6 to 10 hours at 30° C. proved to be avirulent when inoculated 1 or 10 days after such desiccation. VI. Action of Ultra-violet Light.^ We proceeded in the followmg manner. A homogeneous emulsion of virulent brain was centrifugaUzed for 5 minutes. The supernatant riuid was carefully pipetted into a small quartz flask and exposed for 5 minutes to the rays from a mercury arc. Two mercury vapour lamps (K.B.B. type, 25 amperes, 210 volts D.C.) were employed 8 inches distant. The flasks were slowly rotated during the exposure so that a fresh thin film of fluid was constantly exposed to the lamp. The flasks dipped periodically into cold water in a trough to prevent overheating during the experiment. The irradiated fluid was inocu- lated into the brain of a rabbit. At the same time a portion of the non-irradiated emulsion was inoculated intracerebrally into two rabbits, which served as controls. The protocols of these experiments are recorded on page 21. The rabbit inoculated intracerebrally with the virus which had been subjected to the rays of the mercury arc, did not show any symptoms during three months, wliile the controls died after 23 and 34 days respectively, showing that the virus subjected to the action of ultra-violet rays (radiations of wave-lengths 5,720-2,320 A.U.) is killed in a maximum of 5 minutes. Kaljbit 1G3a was reinoculated, along with a control rabbit, Xo. 25a, with fresh passage virus 86 days later and died on the ' Wc are indfbtml to Dr. Kidinow of the Depart menf of Applied Physiology ( Katiunal Institute for Medical R<-Bearch) for his collaboration in these exp«) „ 5.9.27. 2,750 „ 11.9.27. 2,740 „ 15.9.27. 2,800 „ 22.9.27. 2,720 „ 6.10.27. 2,740 „ 12.10.27. 2,800 „ 21.10.27. 2,780 „ „ 15.8.27. 1,800 gms. Nonnal. 23.8.27. 1,750 ., 30.8.27. 1,650 „ 5.9.27. 1,800 „ 11.9.27. 1,950 „ 15.9.27. 2,010 „ 22.9.27. 1,980 „ 6.10.27. 2,000 „ 12.10.27. 2,000 „ 21.10.27. 2,010 „ 15.8.27. 2,(60 gms. Normal. 23.8.27. 2,150 „ 30.8.27. 1,900 „ 5.9.27. 1,780 gni.s. Disease commencing. 11.9.27. 1,550 gms. Typical dUease. 14.9.27. Found dead 30th day. Typical microscopic lesions in central nervous system. Conclusion. The conclusion arrived at is that formalin in a con- centration of 0-2 per cent, inactivates the virus after 18 hours' contact at room temperature. X. Culture. All attempts at cultivation of the virus liavc remained ncfjative up to the present. XI. Summary. From the observations of the authors quoted and our own, it appears that the virus of Boma disease possesses the properties common to those of vaccinia, herpes, rabies, and poliomyelitis whicli Levaditi has grouped together under the name ' ectodernioses neurotropes*. Under favourable conditions it filters through bacteria-proof filters, ENZOOTIC ENCEPHALO-MYELITIS 23 although most of the virus is held back, and through a collodion ultrafilter which will allow colloidal particles of large dimensions to pass. The infectivity of the supernatant fluid cannot be removed by centrifugalization for 15 minutes at 5,400 revs, per minute. It is resistant to the action of glycerine, but sensitive to desiccation, ultra- violet hght and heat. It is destroyed by ether, chloroform, and for- mahn. It has not been propagated outside the body. 3. TRANSMISSION OF EQUINE STRAIN TO RABBIT AND FROM RABBIT TO SHEEP, AND VICE VERSA Moussu (1926) inoculated an emulsion of the brain of a rabbit pre- viously infected with the virus from a horse into the anterior chamber of the eye of a horse. The animal developed symptoms 3 days after the inoculation and died in 8 days. The lesions found in the brain were very intense, infiltrative, and haemorrhagic. A rabbit inoculated with an emulsion from the brain of this horse died 4 days later.i This same author failed to infect horses with virulent material from rabbits by subcutaneous inoculation or per os. Zwick and his col- laborators (1926) inoculated a horse intracerebrally with the brain of a rabbit suffering from experimental Borna disease. The virus had been passaged in this species of animal nine times. The horse fell ill 53 days after the inoculation. For 11 days it showed the typical symptoms of encephalo-myehtis and death followed 64 days after the inoculation. The lesions of the nervous system were characteristic of Borna disease. The intranuclear corpuscles of Joest-Degen were demonstrated in the ganghon cells of the Ammon's horn. We have referred previously to the experiments of Beck and Froh- bose (1926), Moussu (1926), Miessner (1926), and Ernst and Hahn (1927), wliich showed that the virus of encephalo-myelitis originating from sheep can be transmitted to rabbits by experimental inocula- tion. Moussu and Marchaud (1924) succeeded in passing the disease from sheep to sheep. Zwick and his collaborators (1926) failed to transmit the disease to adult sheep by intracerebral inoculation with a strain derived from a horse and passed through rabbits. Using the same strain of virus they succeeded, however, in conferring the disease on young lambs. Death followed 88 days after the inoculation and typical cerebral lesions were revealed. Direct inoculation from horse to lamb was also successful. The lamb showed characteristic symptoms and died 92 days after infec- tion. Inoculation of the brain of this lamb to a rabbit gave a positive result, but inoculation of the cord gave a negative result. Beck and Frohbose (1926) did not succeed in infecting the horse by the intra- cerebral route with virus from sheep dead from the spontaneous ' The experimenta of Moussu and Marchand have been criticized since his inocu- lated animals succumbed very early, and also because the lesions of the brain were surprisingly acute when compared with those found in the classical disease occurring spontaneously or in animals infected with the viruses isolated by the German school. We had the intention of comparing their strain with those of Zwick and Miessner, but Moussu has informed us that his strain is not now available. 24 BOEXA DISEASE AND disease. On the other hand, they succeeded in infecting sheep with virus obtained from horses. From the foregoing resume of the hterature the following conclu- sions may be drawn: (1) The virus originating from horses passaged through rabbits can be transmitted back to the horse. (2) The virus taken directly from the horse or subsequently pas- saged through rabbits is pathogenic for lambs. (3) Attempts at transmitting the disease from sheep to horses have so far been unsuccessful. 4. EXPEEIMENTAL DISEASE IN THE EABBIT I. Experimental Transmission of the Disease to the Rabbit Moussu (1926) inoculated an emulsion of the brain of a horse dead of encephalo-m5-ehtis into the anterior chamber of the eye of the rabbit. In one experiment three animals were inoculated by this route. One died on the 9th day; the two others survived; the rabbit which died constituted the head of the series of passages that the author continued until he obtained a 'fixed' virus which killed the rabbits infected by the intraocular route in 4 to 6 days. In another experiment using similar material one out of live animals inoculated intraocularly died 11 days after receiving the injection; the other four survived. In the majority of cases a marked excitability was a characteristic symptom. Moussu states, however, that certain rabbits die 'following an infection with a slower evolution, lasting more than a fortnight'. Zwick inoculated rabbits intracerebrally with the cerebral sub- stance (Ammon's horn, caudate nucleus, and cerebral cortex) taken from the brain of a horse dead of Borna disease. He observed typical symptoms of the disease after a period of about 4 weeks, and the lesions were analogous to those described previously in the horse. Passage from rabbit to rabbit was effected, the period of incubation after inoculation being about 3 weeks. Zwick and his collaborators (1926) succeeded in infecting rabbits with the virus from 16 out of 21 cases of horses dead of Borna disease which was verified histologically. The incubation period of the disease in such rabbits infected by the intracerebral route was from 3 to 4 weeks. Death ensued 8 to 14 days after the appearance of the characteristic symptoms. Beck and Frohbose (1926) also infected rabbits with the virus from horses and sheep. Miessner (1926) with sheep virus, and Ernst and Hahn (1927) with viruses from horses, sheep, and cattle. II. Authors' Observations. The virus of encephalo-myelitis of equine or ovine origin isolated by tlie (terman workers does not become 'fixed' when passaged through rabbits. The period of incubation varies from 15 to 50 days, and w(' have also observed recurrent forms of the disease in rabbits. ENZOOTIC ENCEPHALO-MYELITIS 25 We have inoculated more than 200 rabbits by the intracerebral route with the virus originating from horses or sheep. In Table I the period of incubation and the time between the onset of the disease and death is recorded for a total of 50 rabbits used for passaging a virus originally obtained from a horse and sent to us by Professor Zwick. Table I. Number Commence- Duration of Weight in ment of of the Lesions in rabbit. gms. disease. disease. 9 Death. C.N.S. 25 2,160 21st day 14 days 35th day Intense. 22 2,000 20tli „ 4 „ 24th „ Positive. 24 1,420 21st „ 19 „ 40th „ ,, IOd 2,500 27th „ 10 „ 37th „ Intense. llD 1,000 17th „ 10 „ 27th „ ,, *93a 1,520 7th „ 1 „ 8th „ Slight.' 68a 1,150 20th „ 5 „ 25th „ Ditense. 50a 1,620 40th „ 13 „ 53rd „ ,, 77a 1,570 38th „ 10 „ 48th „ ,, 80 1,360 21st „ 7 „ 28th „ ,, 5lA 1,350 20th „ 8 „ 28th „ ,, 34 2,000 43rd „ 14 „ 57th „ „ 298 820 25th „ 14 „ 39th „ ,, 275 2,100 25th „ 7 „ 32nd „ ,, 202 1,050 29th „ 3 „ 32nd „ „ 282 1.040 27th „ 4 „ 31at „ ,, 244 1,200 33rd „ 12 „ 45th „ „ 261 1,280 23rd „ 8 „ 31st „ „ 232 1,340 20th „ 11 „ 31st „ ,, 219 1,820 16th „ 5 „ 21st „ „ 295 1,300 9th „ 10 „ 19th „ Very intense. 211 1,580 31st „ 11 „ 42nd „ j^ 212 1,400 31st „ 13 „ 44th „ ^, 220 1,500 31st „ 8 „ 39th „ ,, 222 1,540 35th „ 13 „ 48th „ ,, 224 1,150 32nd „ 13 „ 45th „ ,, 226 1,150 24th „ 4 „ 28th „ Positive. 70 1,700 20th „ 2 ,, 22nd „ Intense. 56 1,400 20th „ 6 „ 26th „ ,, 61 1,500 24th „ 6 „ 30th „ 251 1,280 18th „ 9 „ 27th „ 237 1,680 18th „ 13 „ 31st „ 255 1,920 35th „ 7 „ 42nd „ 256 1,860 32nd „ 15 „ 47th „ 30 1,120 28th „ 8 „ 36th „ 44 1,350 26th „ 7 „ 33rd „ 58 1,000 27th „ 7 „ 34 th „ Positive. *291 950 No sympt. — 15th „ Slight.' 85b 1,500 32nd „ 6 „ 38th „ Very intense. *43 1,020 No svmpt. — 12th „ Slight. 60 1,800 30tii „ 7 „ 37th „ Intense. 62 1,610 22nd „ 17 „ 39th „ ,^ 271 1,220 31st „ 10 „ 41st „ „ •80 820 No sympt. — 7th „ Slight. 269 2,200 19th „ 2 „ 21st „ Positive. •775 1,780 No sympt. — 13th „ Positive.' 296 2,900 28th „ 7 „ 33rd „ Intense. 277 2,100 13th „ 11 ,. 24th „ „ 78s 1,700 24th „ 8 „ 32nd „ ^^ 116a 1,700 18th „ 4 „ 22nd „ " ' When the brain of these rabbits was passaged, positive results were obtained. Rabbits inoculated with passage of virus from the brain of Rabbit No. 93a died in 26 BOEXA DISEASE AND Of 50 rabbits inoculated in the brain : 23 died between 21 and 33 days after inoculation. 6 died in less than 21 days. 21 died in more than 33 days. Only exceptionally did the rabbit die in less than 3 weeks when infected by the intracerebral route. The detailed histopathological study of each case showed that the rabbits dead 7 to 8 days after inoculation had minimal infiltrative lesions in the central nervous system. The presence of virus in the brain was proved b}' subsequent passage and in all cases complete autopsies were made to exclude the possibihty of death from other causes. The animals which died between 21 days and 57 days presented the characteristic lesions in the nervous system, which are described in full in the chapter dealing with the histopathology of the disease. The intensity of the lesions was not in direct relationship with the duration of the malady. As has also been observed by Zwick the incubation period was longer in larger animals. Generally, rabbits weighing less than 1,500 gms. were more susceptible to infection than older rabbits. Excluding the five animals in Table I marked with an asterisk, all of which died in 15 days or under, the average time which elapsed between the intracerebral inoculation and the death of the animals in our experiments was 20 daj's in rabbits of less than 1,500 gms. and 86 days in rabbits over this weight at the time of infection. Table II. Number Commence- of Weight in ment of Duration Lesions in rtMit. gnu. disease. of disease. Death. C.NJS. 164 1,220 21st day 8 days 29th day Intense. 18 1,240 26th „ 9 „■ 35th .. 246 1,380 28th „ 10 „ 38th ., „ 234 1,420 7th „ — 7th „ Slight. 243 1.420 20th „ 2 „ 22nd „ Intense. 235 1,500 22nd „ 9 „ 33rd „ „ 262 2,220 40th „ 8 „ 48th „ „ -. —o _. 1 k \ ■ 1/ \ ' i / \ V \l V 1 LEUCOCYTES \ DIED \ o , 1 1 1 1 1 1 b 10 14 18 22 26 10 14 18 22 26 ^ 3 inoculated intracerebrally was taken daily at the same hour. Death takes place in coma — the temperature being hypothermic (35° C- 34° C. : see Chart II). IV. Routes by which Rabbits can be Infected. Intracerebral. Intracerebral inoculation produces the disease in a constant manner, followed by death. Intratliecal. Beck (1925) infected by introducing virus intrathecally, and we have also succeeded in infecting rabbits by this route. Experiment 1 . Rabbit 207, weighing 780 gms. was inoculated intra- thecally in the lumbar region with 0-5 c.cm. of a virulent emulsion of brain diluted 1 : 10 in physiological saline. Forty- three days 30 BORXA DISEASE AND after the inoculation the condition of the animal was such that when taken out of the cage it walked with its legs spread out from the body. Paresis of the hiiid quarters was well marked, and increased gradually. The animal wasted and died 66 days after the inoculation. Examination of the brain revealed the presence of characteristic lesions, and a passage of this brain to a healthy rabbit gave a positive result. Sciatic Nerve. Introduction of several drops of a virulent emulsion of the virus into the sciatic nerve did not infect animals with encephalo-mj'eUtis in the experiments of Moussu (1926), and Zwick and his collaborators (1926). We have, however, succeeded several times in producing Boma disease in rabbits inoculated by this route. Experiment 2. The right sciatic nerve of Eabbit 208, weighing 850 gms., was exposed by incision and 2 or 3 drops of a virulent emulsion of the brain of a rabbit infected with encephalo-myehtis was injected into the substance of the nerve-trimk. The point of intro- duction of the needle was seared to prevent the exit of fluid into the surrounding tissues. The operation was carried out aseptically and the incision healed by first intention. The animal showed no morbid symptoms for 35 days and put on weight, reaching 1,350 gms. Subsequently it became prostrate, wasted progressively, and showed marked inco-ordination, which became accentuated later; the animal died on the 48th day. The brain was proved to be bacteriologically sterile, and no lesions were found which might ser^-e to explain the cause of death, other than those in the central and peripheral nervous system. These were of an intense character, and were found in the brain, in the spinal cord (cervical, thoracic, dorsal, and lumbar regions), and also in the inoculated nerve. Emulsions from the brain and also the dorso-lumbar part of the spinal cord were inoculated into the brain of fresh rabbits. These inoculations produced the disease, showing that the virus was present both in the brain and cord of rabbits inoculated into a peripheral ner%'e. Babbit 270 was also inoculated into the sciatic nerve by the same method as recorded above for Rabbit 208. This rabbit (270) showed, 65 days after the inoculation, paralysis of the leg into the sciatic nerve of which virus had been inoculated. Paralysis of the hind quarters followed, with grinding of the teeth, and other typical symptoms. The animal was found dead on the 78th day. Lesions wore demon- strated throughout the nervous system (brain, mesencephalon, cord in all regions, inoculated nerve, the sciatic of the opposite side (non- inoculated), as well as in the brachial nerves). The detailed description of these lesions will be given later. Rabbit 27t> was inoculated into the right sciatic with the same technique as before. The leg on the side of inoculated nerve became useless after 28 days, and the animal died 8 days later with lesions in the central and peripheral nervous system. ENZOOTIC ENCEPHALO-MYELITIS 31 Anterior Chamber of Eye. Moussu (1926) and also Zwick (1926) have shown that it is possible to infect the rabbit by inoculation of a virulent emulsion of the brain of the horse into the anterior chamber of the eye, and we have con- firmed the possibility of infection by this route. Rabbit 206 received several drops of the supernatant fluid from a virulent emulsion of brain into the anterior chamber. The point of inoculation was carefully seared. During the period immediately fol- lowing the inoculation a coagulum of fibrin could be seen in the eye, but this was absorbed later. The animal died 23 days after the inocu- lation, and lesions characteristic of Borna disease were foimd in the central nervous system; these, however, were not very acute. The control rabbit inoculated intracerebrally with the same emul- sion died in the average time with well-marked lesions in the central nervous system. Rabbits 86s and 89s inoculated in the anterior chamber with an emulsion containing virus fell ill 29 and 36 days respectively after infection, and died on the 34th and 51st day with typical symptoms in the central nervous system. The control rabbit (intracerebral route) died 37 days after inoculation. A fourth rabbit inoculated intraocularly with the virus survived without having shown any symptoms. Thus, of four rabbits inoculated in the anterior chamber three became infected and died of the disease, while the fourth showed no morbid symptoms and survived. These results are in accordance with those of Zwick who had five positive results in six attempts to infect rabbits by the intraocular route. No macroscopic modification of the cornea followed the introduc- tion of the virus, but microscopically there was slight infiltration with lymphocytes between the corneal laminae. In one case an interstitial infiltration with mononuclear cells of the optic nerve was found. Corneal Scarification. Zwick (1926) has stated thatmfection by corneal scarification causes the disease only rarely. We have inoculated four rabbits by scarifica- tion of the cornea; none became infected, nor was there any visible keratitis. However, one of the rabbits (Rabbit 81s) which was sub- sequently reinoculated intracerebrally with a virulent emulsion of brain 110 days later remained well, whereas a control rabbit which had received a cerebral inoculation with the same emulsion died 48 days later after showing typical symptoms of the disease, and with the characteristic lesions. The corneal inoculation may have rendered the rabbit refractory to infection by the intracerebral route. Conjunctival Sac. Instillation of a virulent emulsion into the conjunctival sac of the eye produced no effect. Nasal Mucosa. The nasal mucosa appears to be a possible portal of entry of the virus, and Joest (1927) has suggested that natural contagion in the 32 BOENA DISEASE AND horse is effected by this route. The results of attempts by Zwick (1926), Beck and Frohbose (1926) to infect rabbits by this route have, however, been inconstant. Scarified Skin. Zwick apphed a virulent emulsion of brain to the scarified skin without result. Our results ^ confirm those of Zwick. As, however, the experiments of Flexner and Amos (1917) with pohomyeHtis, and of Levaditi and Nicolau (1922, 1923) with herpes and neurovaccinia show that previous injection of substances hke physiological saline, bouillon, or normal serum into the brain may increase susceptibiUty, we introduced physiological saline either into the brain or intra- thecally in rabbits which had received an apphcation of the virus on to the depilated, shaved, and scarified skin. The animals prepared by the inoculation of sahne intrathecaUy did not show any symptoms of the disease, while those which had been subjected to an irritation of the brain \vith saline subsequently contracted the disease, and died. Typical lesions were found in the central nervous system, and passage of the brain to new rabbits by the mtracerebral route gave positive results. This experiment shows that when the nervous system is in a state of special receptivity due to the diminution of the normal power of defence, infection can take place from the skin. The virus probably reached the brain by way of the intercostal nerves, thus being protected from the action of leucocytes circulating in the blood. This interpretation is supported by the following experiments. Four rabbits were inoculated with 1-5 c.cms. of a centrifugalized emulsion of virulent brain into the marginal vein of the ear. Two of the rabbits inoculated intravenously received simultaneously 0-3 c.cm. of physio- logical saline into the brain, but neither of these animals contracted the disease, nor did the two which received only the intravenous inoculation. Two months later the immunity of these four rabbits was tested by intracerebral inoculation, and all proved susceptible to infection. The virulence of tiie enmlsion used to infect the four rabbits intravenously was proved by the intracerebral inoculation of two controls. So that it would appear that the virus was destroyed in the blood-stream, or in the tissues before it reached the brain. Our further attempts to infect by the intravenous route gave us negative results, but Zwick (1926) succeeded exceptionally when 4 intravenous injections were given at intervals. Positive results have also been obtained by Ernst and Hahn (1927). Usually, however, they found that repeated inoculations by the intravenous route instead of conferring the malady produced solid resistance. This will be discussed in the chapter dealing with immunitj'. Subcutaneous. Subcutaneous inoculation may, exceptionally, lead to a fatal en- cephalo-myelitis. Zwick and his collaborators (1926), who employed repeated injections of the virus, produced the disease with a greater ' At no time did the skin Dhow any macroscopic changes which cuuld be attributed to the viruB. ENZOOTIC EXCEPHALO-MYELITIS 33 frequency. They obtained similar results by inoculation of the virus intraperitonealhj; intramuscular injections with tlie virus did not confer the disease m their experiments. Intratestwular. According to the latter investigatoi-s the introduction of virus by the iuti-atesticular route did not produce infection in rabbits, but in our experiments it has done so. Babbit 209, weighing 1,850 gms., was inoculated mider anaesthesia into both testicles \\ith a virulent emulsion. of the brains of four rabbits dead of experimental Borna. The dilution of the emulsion of brain substance in physiological saline was 1 : 20. During a period of 43 daj's the animal showed no morbid symptoms and its weight increased to 2, 540 gms.; then, without other symptoms, wasting commenced; 19 days later the weight had fallen to 1,860 gms. (680 gms. loss), and inco-ordination with shght paresis of the hind quarters was noticed. Paresis became accentuated and other symptoms charac- teristic of the disease became manifest. On the 71st day after the inoculation. 20 days after the first loss of weight was recorded and 8 days after the first chnical symptom, the animal died, weighing only 1.4S0 gms. The lesions found in the central nerv^ous system were characteristic and were especially intense in the lumbar region. The topogi'aphy of the lesions in the cord indicated that the virus had spread from the point of inoculation to the bram through the cord centripetally. The intranuclear inclusions of Joest-Degeu were found. The passage of the brain and cord of Rabbit 209 to fresh animals gave positive results, indicating the presence of virus in both. The control rabbit inoculated intracerebrally with the emulsion of brain which served to infect Rabbit 209 died 32 days after inoculation. Rabbit 289. weighing 1,720 gms., was inoculated into the right testicle with a \'irulent emulsion of brain. During 60 days it put on weight, reaching 2.320 gms. Tliis weight was maintained for 14 days, when wasting began. On the 90th day after inoculation paresis of the hind quarters was observed. The animal died 105 days after inocula- tion. Rabbit 273 inoculated into the right testicle at the same time showed no symptoms and sm^vived, while the control rabbit inoculated intra- cerebrally died after 37 days with the typical symptoms and lesions characteristic of the disease. Intratracheal. We have made two unsuccessful attempts to infect rabbits by intratracheal inoculation. In the first attempt two rabbits received each 0-3 c.cm. of a thick emulsion of brain containing virus into the trachea, which had been exposed by incision; both these rabbits sm-- vived without having shown any symptoms, while the control which had received an intracerebral inoculation with the same virus suc- cumbed to the infection. In the second experiment four j'oung rabbits, between 670 and 860 gms., were inoculated. Each received 0-5 c.cm. of a thick emulsion of the brain of a rabbit, dead of Borna disease, 6409 „ 34 BOENA DISEASE AND diluted 1 : 5. Kept under observation more than six months they maintained their normal state of health, more than doubling their weight. The controls of this experiment inoculated intracerebrally died of a typical encephalo-myehtis on the 4'2nd and iith day after injection. Per Os. Attempts at infecting rabbits per os are of special interest for the interpretation of natural infection in horses, cattle, and sheep. Zwick and his collaborators (1926) succeeded in infecting rabbits by mixing virulent brain with the food. He refers to this as infection by the intestinal route, but as the virus was administered by the mouth with the food, the pre-existence of small traumatic lesions in the mouth might permit the implantation of the virus. As infection by the nasal mucosa has been shown to be possil)le, one cannot exclude the possi- biUty that infection took place by the buccal mucous membrane, especially when one considers the existence of nervous tissue imme- diately below the mucous membrane covering the tongue (Manouehan and Viala, 1926). Supposing the virus to have been implanted in such nervous tissue, it is quite easy to conceive how it might ultimately reach the brain. Attempts to infect rabbits by cohabitation have been unsuccessful. 5. AUTHORS EXPERDIENTS ON THE TRANSMISSION OP THE DISEASE TO MONKEYS {MACACUS RHESUS), AND SYMPTOMS OCCURRING IN THESE ANIMALS Monkey >/. 1. A fine specimen of Macacus rhesus weighing 3,800 gms. was kept under observation during 16 days prior to inoculation. The animal's temperature varied very little. On 15.3.27 it was inoculated intracerebrally under anaesthesia with 1-5 c.cms. of a vindent emulsion of the brain of a rabbit diluted 1 : 5. Two control rabbits were inoculated intracerebrally at the same time as the monkey; they developed typical symptoms on the 28th and 31st day, and died of Borna disease on the 42nd and 44th day. Between 15.S.27 and 11.5.27, a period of 57 days, the monkey showed no symptoms and the tenij)erature remained normal. 15.3.27. Weight 3,800 gms. Temperature 38-9' C. Received inoculation w-ith the brain emulsion from Rabbit 275. 11.5.27. Fifty-seven days after inoculation the monkey appeared depressed. Tem- perature 38-4° C Slight diarrhoea. 12.5.27. Same condition. 14.5.27. Temperature 38-5 C. No diarrhoea. L<'»« Uvely than usual, appearetl to prefer to remain with the back to the light (photophobia ?). 16.5.27. Condition unchanged. 16.5.27. Photophobia well marked. The monkey hid its head under the straw of the cage and would not move when disturbed. It allowed itself to Im? caught easily and dcfendeartially. 25.7.27. Eye nearly completely open. Animal still aggmisive, uttered cries from time to time, paralysis less noticeable. ENZOOTIC ENCEPHALO-MYELITIS 37 28.7.27. Eye appeared normal, and the condition of the monkey practically normal. 1.8.27. Ptosis of the right eyeUd. Slight paresis of the hind quarters, which, however, was not sufficient to prevent the monkey from climbing. It became fatigued easily, however, and remained in a corner of the cage crying out from time to time. 3.8.27. Paralysis of the right side of the face (see Fig. 5) was observed. Animal could .still run and climb. 5.8.27. Same condition. Weight 3,170 gms. 8.8.27. Paralysis of face diminished. Animal irascible. 12.8.27. Spasmodic contractions of muscular groups of the back and shoulders were the only signs of the animal being other than normal. 13.8.27. Left eye deviated to the internal canthus. Tendency to remain hunched up. 14.8.27. Animal decidedly ill. Head hanging over on to right shoulder : internal strabismus still present and left pupil dilated. Right eye mobile. Pupil of this eye reacted to light. Nystagmus present. The animal had periods of excitement. 15.8.27. Monkey uttering cries with a low feeble raucous voice. Mouth opened after shomng dragging to the right side. Internal strabismus on the left side, pupil on this side also dilated. Nystagmus exacerbated. Paralysis of muscles of left shoulder, head falling over on to the right shoulder. 17.8.27 and 21.8.27. Condition unchanged. 27.8.27. Until 27.8.27 animal appeared normal. On this date the animal had complete aphonia (probably paralysis of the recurrent nerve). The head hung over the right side and the monkey showed signs of cerebral disorder. Strabismus was less, face was drawn to the right side, and there was frequent spasmodic contraction of the facial muscles ('tic). The pupils were unequal in size. The animal carried out movements of mastication without the teeth coming in apposition, for this reason it fed with difficulty. Deglutition was not carried out easily, and the animal appeared to have difficulty in orientation. Taken out of the cage it could not co-ordinate its movements in the direction desired. 29.8. 27. Weight 2,900 gms. Animal still made movements of mastication con- tinually, and twitching of the muscles of the mouth was present. There was internal strabismus, as weU as aphonia and weakness of the muscles of the neck on the left side. The monkey remained hunched up, or had periods of excitement which were increased by noises, movements, &c. 31.8.27. Condition unchanged. 2.9.27. Weight 2,820 gms. Spastic contractions of divers muscular groups were produced by noises. The tongue was drawn to the right side. The animal fed no more owing to the impossibility of swallowing. 'Tic' of the mouth persisted, also the strabismus and the inequahty of the pupils. 4.9.27. The left eye appeared practically normal and the pupils equal in size. The animal, however, still trembled at times and bit at imaginary objects; frequent 'tic' was observed with aphonia. The animal, how- ever, fed well. 9.9.27. The condition of the animal had not changed. 16.9.27. The cry appeared more normal, ocular disturbances were absent, although 'tic' and champing of the jaws was still observed. The head was hung over the right shoulder, and the animal showed increased salivation. 20.9.27. The condition of the animal was unchanged; to a certain degree the syndrome in this monkey might be compared to that exhibited by a man affected with post-encephaUtic Parkinsonism. 22.9.27. Same condition. Weight 3,000 gms. 5.10.27. Up to this date the monkey remained in a similar state, this being the 130th day since the inoculation. At this time the animal was inoculated with the virus of polio-myelitis. The result is given in the chapter dealing with immunitv. 38 BOENA DISEASE AND These three monkeys showed three different forms of the disease. In all three cases the incubation period was very long, but the dura- tion of the morbid symptoms varied. Monkey 3 had an acute attack which lasted only 2 days. In Monkey 1 the evolution was slower, the time between the onset of symptoms and death being 16 daj's. Monkey 2 had recurrent attacks during a period of 97 days (see sub- sequent history, p. 80). The virus after passage through monkeys liad not lost its \irulence for the rabbit or guinea-pig. In the chapter dealing with histopathology the lesions in the nervous sytem will be described in full. We may, however, anticipate here by the statement that we found lesions of an intense character, in the brain, and of a more discrete character in the cord. Lesions were also found in the spinal ganglia, posterior nerve-roots, and peri- pheral nerves. Both clinically and pathologically the disease may be described as an encephalo-myelitis complicated with a ganglio- radiculitis and peripheral neuritis. The virus when introduced into the brain evidently spreads not only to the cord, but also to the peripheral nerves, for not only have we found lesions in these, but we have also been able to demonstrate the presence of virus. Discussion. The pathogenicity of the virus for the monkey, and the clinical features presented in this animal, raise the question of the relation between enzootic encephalo-myelitis of domestic animals and polio myelitis in man. There are marked resemblances in the clinical aspects as well as in the alterations in the cord and spinal ganglia in the two diseases. The former virus is, however, pathogenic for the rabbit, while the latter is generally considered not to be so. The incubation period of experimental encephalo-myeUtis in the monkey is longer than in the disease produced by the virus of poho-myelitis. Another question is whether the virus of encephalo-myelitis is pathogenic for man, and if such is the case, whether some luunan disease of the nervous system at present of unknown origin may possibly be due to it. It is a common observation in clinical medicine that exposure to cold may determine an attack of facial neuritis or sciatica. Conceivably, such attacks might be the expression of an unrecognized infection by some virus, akin to enzootic encephalo- myelitis, which in the lirst place caused only shght. if any, general symptoms of disease and then proceeded down into the peripheral nerves, where it remained latent until the added factor of cold deter- mined the local incidence of paralysis or pain. Similar views may be argued with regard to herpes zoster, recurrent herpes, or the so-called peripheral forms of epidemic encephalitis. An analogous action of cold was recorded liy Pasteur, when certain rabbits that were resistant to the inoculation of an atteiuiated ral)ies virus, at once showed paralytic symptoms after exposure to severe cold. The curious epidemic at Lille, studied i)y David and Dekester ENZOOTIC ENCEPHALO-MYELITIS 39 (1926), has suggested that a certain form of sciatica, at least, is an infectious disease; and there are many human cases on record of myelitis associated with peripheral neuritis, apparently of a contagious type, in some of which, as in the instance of the child described by Pehu and Dechaume (1927), there were in the peripheral nerves in- flammatory lesions closely resembling those described by us in monkeys and rabbits infected with Borna disease. We give below a resume ot Pehu and Dechaume"s case in some detail because the symptoms resemble in certain respects those which our Monkey M. 2 showed after inoculation with the virus of Borna disease. The child, 20 months old, was in perfect health up to the day when it showed some lassitude three months before going to hospital. The temperature did not exceed 36-5° C. Two months later it could no longer walk and had lost power in the arms. At the time of entering the hospital it showed flaccidity and paralysis in the lower extremities without Babinski's sign. There was also slight paresis of one arm. The following day the child collapsed although not losing conscious- ness. There were no cerebral symptoms, nor vomiting, neither did somnolence exist, but the pulse was rapid and irregular. The condi- tion lasted 11 days and the child died suddenly without convulsions. The case was diagnosed as a peripheral form of epidemic encephalitis, referred to as pseudo-myelitic. On autopsy there were no macroscopic changes. On microscopic examination of sections, discrete lesions, for the most part exudative, were found in the cord, especially in the lumbar region. Perivascular infiltrations occurred in the brain. No neuronophagia was recorded, and the spinal ganglia were not examined. The lesions found in sec- tions of the median nerves, sciatic nerve, and posterior tibial nerves were comparable to those described by us in the sciatic and brachial nerves of rabbits and monkeys inoculated intracerebrally with the virus of Borna disease (see pages 61 and 67). Pehu and Dechaume suggested that the presence of lesions in the peripheral nerves might be coexistent with the presence of virus. In the case of Borna disease we have proved that the presence of virus is coexistent with the existence of lesions in the peripheral nerves (see page 45). 6. PATHOGENICITY OF THE VIRUS OF ENZOOTIC EN- CEPHALO-MYELITIS FOR THE GUINEA-PIG, RAT, MOUSE, AND FOWL I. Guinea-pig. The introduction of virus intracerebrally into guinea-pigs may pro- duce the disease. The incubation period varies in individual cases and death is inconstant. Zwick and his collaborators (1926) were the first to transmit the disease to guinea-pigs, and to make passages in series from brain to brain. Some guinea-pigs proved to be resistant to infec- tion. According to their experiments death followed infection in 40 BORNA DISEASE AND from 3 weeks to 13 months. They also succeeded in infecting rabbits with the virus passaged through guinea-pigs. In Table III we give the period of incubation and the duration of the disease in a batch of guinea-pigs which in our experiments proved to be susceptible to the virus inoculated intracranially. Table III. No. of guinea- Weight in Commencement Lesions in pig. gms. of disease. Death. brain. 85E 470 73rd day 83rd day Intense. 84k 400 107th „ 132nd ,. .. 94e 500 23rd .. 114th .. 95k 500 58th ., 65th ,. Discrete. 93k 480 40th ., .58th .. .\verai^. 98k 450 57th .. 64th ,. 99k 580 18th „ 19th „ Intense. 1()0k 560 58th „ 182nd „ We have also made experiments to determine the relative suscepti- biUty of guinea-pigs to infection. Forty-five guinea-pigs of about the same size (400-GOO gms.) were divided into tliree lots of 15. Lot A were injected with an emulsion of the brain of a rabbit dead of Boma disease diluted 1 : 10. Lot B were injected with the same emulsion diluted 1 : 100. Lot C were inoculated with the emulsion diluted 1 : 1.000. The results were as follows : In Lot A all the guinea-pigs died after showing typical symptoms of the disease. 52, 58. 74. 133, 134, 139, 139, 141, 141, 143. 148, 150, 153, 15(5. and 170 days after the inoculation. Lesions characteristic of enzootic encephalo-myelitis were found in sections of the brain and spinal cord of these animals; moreover, the corpuscles of Joest- Degen were demonstrated. Tablk IV. Lot A. Dilution of emulsion of brain inocu- A'o. of Animal lated. gutnea-pig. died. 1:10 lA 2a 3a 4a 62nd day SSth „ 74th „ ISSrd .. Intense lesioiu in rentral nervnuii Nyato n. 5a 134th .. Very intense le«iiin in C'.N.S. Ha 139th .. Discretei lesions in C'.N.S. 7a 139tb „ Intense lesions in C.N.S. 8a 14Ut „ P, M 0A 14l8t ., Mild lesions in C.N.S. 10a 143rd „ Intense lesions in C'.N.S. ■ llA U8th „ M ,. 12a 150th „ 13a 163rd .. Very intense Icoions in C.N.S. 14a 166th ., Intense li«ii>ns in I'.N.S. 15a 170th „ ENZOOTIC ENCEPHALO-MYELITIS 41 In Lot B two of the guinea-pigs succumbed to an intercurrent infection. The thirteen others died 58, 105, 130, 130, 135, 137, 138, 140. 140, 140, 147, 149, and 153 days after infection. Table V. LotB. Dilution of emulsion of brain inocu- So. of A n imal lated. gmnea-pig. died. Observations. 1:100 IB 58th day Intense lesions in central nervous system. jj 2b 105th „ Slight lesions in C.N.S. ,, 3b 130th „ Very intense lesions in C.N.vS. „ 4b 1.30th „ Intense lesions in C.N.S. OB 135th „ ». ,» „ 6b 137th „ i» »» J, 7b 138th „ „ » „ 8b 140th „ ,, n ,j 9b 140th „ Slight lesions in C.N.S. „ 10b 140th „ Intense lesions in C.N.S. „ 11b 147th „ .. »» J, 12b 149th „ » „ „ 13b 153rd „ ., ,. „ 14b 5th „ Accidental death. •• 15b 12th „ „ In Lot C five of the animals died of an intercurrent infection, the ten remaining died 70, 90, 96, 101, 140, 141, 141. 147, 149, and 150 days after intracerebral inoculation. Table VI. Lot C. Dilution of emulsion of brain inocu- No. of Animal lated. guinea-pig. died. Observations. 1 : 1,000 Ic 70th day Discrete lesions in central nervous system. 2c 90th „ Intense lesions in C.N.S. 3c 96th „ ,, », 4c 101st „ „ >> , i5c 140th „ ,» ,, J 6c 141st „ >• „ 7c 141st „ ,» .. 8c 147th „ „ ,. 9c 149th „ Very intense lesions in C.N.S. ^ 10c 150th „ », »• ,» lie 5th „ Accidental death. 12c 8th „ .♦ », 13c 8th „ >, „ , 14c 8th „ .. •, 15c nth „ „ As in the case of the guinea-pigs of Lot A, the symptoms in guinea- pigs of Lot B and C were characteristic, and sections made from the 42 BORNA DISEASE AND central nervous system showed the typical changes produced by the virus of Boma disease in other animals. These results, while demonstrating the variation in individual sus ceptibility, also point to the fact that resistance to infection in the guinea-pig is not so marked as it appeared to be from the results obtained by Zwick, since of thirty-eight gvunea-pigs inoculated in our experiments thirty-eight succumbed to the disease (the seven guinea- pigs dead from other causes are not mcluded). The virus passaged through guinea-pigs still preserved its patho- genicity for the rabbit. The disease in the guinea-pig is similar to that of the rabbit. After a variable period the animal appears depressed, there is marked somnolence, and abstention from food. Characteristic nervous symp- toms follow, those indicating affection of the cord being especially well marked. The syndrome is as described in the rabbit. The bind legs become paralysed (Fig. 6) and the fore legs are mvolved later (Fig. 7). The loss of weight is less marked than in the case of the rabbit. From the four following experiments the susceptibility of the guinea-pig after the virus is inoculated would appear to be diminished by a simultaneous inoculation of the same material intramus- cularly. An emulsion of virulent brain originating from a rabbit dead of experimental Boma disease was inoculated into the brain of eight guinea-pigs, and at the same time 1 c.cm. of the same emulsion was inoculated into the quadriceps group of muscles of four of them. The results of these four experiments are tabulated below: Table VII. No. of guinea-pig. <.I5k 97 b Ouinea-pig died. 65th day 13l8t „ Letions in the brain. I. Inoculated into the brain Inoculated into the brain and muscle. Discrete. Intense. II. Inoculated into the brain Inoculated into the brain and muscle «3k 91 B 58th „ 116th „ Of average intensity. III. Inoculated into the brain IniM'ulated into the brain and muscle 99k 9«B 19th day Survived Intense. IV'. Inoculated into the brain Inoculated into the brain 98k 2UUu 64th dav 184th ., Of average intensity. Intense. iiiid muscle This observation is comparablt^ to that of Ernst and Halm (1927), who found that when rabbits inoculated intracercbrally witli viru- lent emulsion received, either at the same time or subsequently, injections of virus into the veins, they did not develop a fatal cnophalitis. ENZOOTIC ENCEPHALO-MYELITIS 43 Attempts at infecting the guinea-pig by intradermal inoculation of an emulsion of virulent brain into the metatarsal pad (following the technique used by Waldmann and Pape (1921) in foot-and-mouth disease, and by Gildemeister and Herzberg (1925) in experimental herpes) did not succeed. The guinea-pigs inoculated varied in weight from 100 gms. to 750 gms., and were kept under observation for over seven months, but no symptoms were seen at any time during this period. II. Eat. Zwick, Seifried, and Witte (1926) infected rats with the virus of Borna disease by intracerebral inoculation. Death supervened 40, 53, and 62 days respectively, after infection. Some rats showed no symptoms and survived. The virus passaged through the rat had not lost its pathogenicity for the rabbit. In- our experiments large rats appeared to be more susceptible to the disease than young animals. Four rats (three old and one yoimg) were inoculated intracerebrally with an emulsion of the brain of rat No. I which died 67 days after infection. (Typical lesions of Borna disease were found m sections of the brain of rat No. 1.) The three large rats died 22, 37, and 74 days respectively after inoculation. They all developed typical symptoms, and sections of the brain showed the presence of characteristic lesions microscopically. The young rats kept under observation for six and a half months remained perfectly normal. The control rabbit inoculated with the same emulsion of brain from Eat 1 died on the 27th day of a typical infection. Subsequently four rats (two large and two small) were inoculated with an emulsion of one of the brains of one of the large rats men- tioned above (that dead on the 37th day). The two older rats died on the 40th and 82nd day after infection, while the two younger animals survived 124 days, succumbing later to an intercurrent infection. In all, we have inoculated twenty-eight rats; of these only the older rats contracted the disease. The symptoms in the rat are similar to those in the guinea-pig. They commence with motor disturbances, inco-ordination, and difB- culty in maintaining equiUbrium. Paralysis, coma, cachexia, and death follow later. Up to the time of writing we have succeeded in making at least four passages in this species. The rats in the series died 67, 37, 82, and 47 days respectively, after inoculation, showing that the course of the disease in the rat is as variable as in the guinea-pig. It would appear that virus passaged through rats when inoculated intra- cerebrally into rabbits produced the disease after a shorter in- cubation period than when the virus was passaged in series through rabbits. In sections of the brain of our experimental rats the corpuscles of Joest-Degen were found. Zwick (1926), however, failed to find them in the brain of rats inoculated with the virus of Borna disease. U BORNA DISEASE AND III. Mouse. We have been able to infect mice by the intracerebral route, but this species of rodent is apparently less susceptible to the infection. As in the rat, age appears to have an important bearing on the sus- ceptibihty of the mouse to the disease. Mice weighing more than 20 gms. generally contracted the disease and died, while smaller mice survived without showing symptoms. In our experiments mice died on the 37th, 52nd, 81st, and 126th day respectively, after inoculation. These mice wasted considerably, walked with tortoise-hke move- ments, and showed other motor disturbances. Typical lesions were demonstrated in the brains of the mice and the intranuclear ' inclu- sions ' of Joest-Degen were present in the Ammon's horn. lY. Fowl. Zwick, Seifried, and Witte (1926) found the fowl to be susceptible to intracerebral inoculation. In one case the incubation period was 37 days, and death followed 15 days later. Passage from fowl to rabbit gave a positive result. 7. ANIMALS WHICH HAVE BEEN FOUND TO BE RESIS- TANT TO INFECTION WITH THE VIRUS OF ENZOOTIC ECEPHALO-MYELITIS I. Dog. According to Zwick, Seifried, and Witte (1926) the dog appears to be resistant to infection with the virus of Boriui disease. This fact obviates up to a certain point confusion with the virus of rabies. A greater number of experiments require to be done, however, before the dog can be definitely classed among the animals resistant to infection. II. Pigeon. These same authors demonstrated that the pigeon is resistant to intracerebral infection with the virus. III. Ferret. We inoculated six ferrets, three young and three adults, by the intracerebral route and kept them under observation for seven months, but no morbid symptoms developed. 8. DISTRIBUTION OF THE VIRUS OF BOUNA DISEASE IN THE ANIMAL BODY I. Passage of Virus through the Placenta. Ernst and Hahn (1927) showed tliat the virus is capable of passing the placenta of the mare and infecting the foetus during intra- ut(!rine hfe. In two cases tht> virus was demonstrated by inoculation ENZOOTIC ENCEPHALO-MYELITIS 45 of rabbits iutracerebrally with the brain of foals born of mothers ill with enzootic encephalo-myeUtis. Further, they demonstrated lesions characteristic of Boma in the sections of the brain of both the foals and the mothers in these cases. II. Distribution of the Virus in Various Organs and Tissues Zwick, Seifried, and Witte (192G) tested four samples of blood from infected rabbits, three samples of blood, two of spleen, two of kidney, and two of Uver, from horses ill from Boma disease, but failed to find the virus. Ernst and Halm (1927), on the other hand, proved the blood to contain the virus during some stages of the illness of a rabbit suffering from Borna disease. Similar apparently contradictory results have been obtained in experimental infections produced by other filterable viruses where the virus may sometimes be found in the blood, e.g. rabies, vaccinia, herpes, and foot-and-mouth disease. Ernst and Hahii (1927) foimd the vitreous body of the eye infective after a rabbit had been inoculated intracerebrally. The virus has also been demonstrated by Zwick and his collaborators (1926) in the submaxillary salivary gland in inoculated rabbits. III. Presence of Virus in the Peripheral Nerves of Eabbits Inoculated Intracerebrally. The present writers have demonstrated the virus in the peripheral nerves of rabbits infected by the intracerebral route in which in- filtrating lesions in the nerve occurred. Experiment 1. A portion of both sciatic nerves taken from 1 cm. below their emergence from the greater sciatic foramen to the popli- teal region was removed aseptically from Rabbit 130a, which died 50 days after intracerebral inoculation. An emulsion of these two portions of sciatic nerve was made in physiological saline and inocu- lated into the brain of Rabbits 213a and 214a. Eahbit 213a. Weight 2,000 gins. 14.7.27. Intracerebral inoculation with emulsion of sciatic nerve. 1.8.27. Animal normal. 2,000 gms. 7.8.27. Animal normal. 15.8.27. Commencement of paresis of the hind quarters. 2,000 gms. 24.8.27. Typical symptoms of the disea.se. 1,600 gms. 29.8.27. Animal very ill. 1,350 gms. 29.8.27. Died the 46th day after the inoculation. Autopsy. All organs macroseopically normal. Cultures from the brain negative. Sections. Intense lesions of a characteristic type in the central nervous system. The corpuscles of .Joest-Degen were also demonstrated. 46 BOENA DISEASE AND Babbit 214a. Weight 2,100 gms. 14.7.27. Intracerebral inoculation with au emulsion of the sciatic nerve. 1.8.27. Normal. 2.250 gms. 7.8.27. Animal normal. 15.8.27. Tj-pical sj-mptoms of the disease. Head depressed; placed on its side the animal showed the characteristic myelitic syndrome. 1,800 gms. 20.8.27. Animal very ill. 1,600 gms. 22.8.27. Found dead 39 days after the inoculation. Autopsy. No lesions in organs. Cultures from the brain negative. Sections. Intense lesions characteristic of Boma disease were demonstrated throughout the central nervous system. Passage. The brain of this rabbit «ri.s passaged to Rabbit 291 x. Babbit 291.K. 24.8.27. Intracerebral inoculation with an emulsion of the brain of Rabbit 214a. 15.9.27. T^-pical symptoms of the disease. 1,590 gms. 21.9.27. Found dead the 28th day. 1,150 gms. Characteristic intense lesions were found in the central nervous system, and the corpuscles of Joest-Degen were demonstrated. The two rabbits died after showing typical symptoms of the disease. Lesions of a characteristic type were demonstrated through- out their central nervous system, and moreover, the virus was demon- strated in their brain. Experiment 2. In a second experiment the virus was sought for also in the brachial nerve of a rabbit which had succumbed 31 days after inoculation into the brain. The nerves were removed aseptically and emulsified in sterile mortars. The emulsions were then inoculated intracerebrally into rabbits. The results of the inoculation are given below. (1) Brachial Xene. Rabbit 36k. Weight 2,870 gms. 15.9.27. Inoculated intracerebrallv with an emulsion of the brachial nerve of Rabbit 252a. 22.9.27. No symptoms. 2,850 gms. 28.9.27. Animal normal. 2,700 grms. 6.10.27. Animal normal. 2,620 gms. 10.10.27. Commencement of paresis. 2,350 gms. 12.10.27. Tj'pical symptoms of the disease. 2,150 gms. 14.10.27. Animal diernmtioii8 in the inucoaa of the Htciinacli of rahhitH dead of Horna disease. We have fimiid these lentiotilar for- mations (see Fiji. !•) in the majority of caaes in which rahlills had a prolonKed jiaralytio phase and a long agonal Ht«ge. llnn an autodigestion of the stomach mucosa. They did nut appcur to have any dilinite struct ure, nor are they s|KH'ilie forex|)i'rinientjil enzootic eucephalo- myelitih, kIiI' <- ue havr found IlKin aJMO In lier|>etic encephalilla and oilier morbid oon- ditionji. ENZOOTIC ENCEPHALO-MYELITIS 53 referred to above, but we bave found tbat tbese are not a specific reaction to tbe virus (Fig. 9). In the larger number of cases examined in detail (more than thirty rabbits) the kidneys showed a marked congestion. This hyperaemia was not limited to the cortical zone, but affected the medullary zone to the same extent. Sections from such kidneys showed that there were small multiple haemorrhages in the region of the glomerulus, as well as in the collecting and con- voluted tubules. These extravasations formed sometimes actual haemorrhagic areas. The epithehum hning the renal tubules was normal, there were no infiltrative processes peri- or intratubular. The condition may be described as renal congestion; not a true nephritis. On microscopic examination the parotid showed occasionally small areas of infiltration composed of lymphocytic elements surrounding certain of the striated canaliculi; at the same time the cytoplasm of certain of the cells of the acini had become oxyphilic when stained by Mann, while the nucleus appeared oedematous and took up an abnormal eccentric position in the cells. Although many sections of the parotid of rabbits were examined, these lesions were found only occasionally. Without presuming that they were produced by the action of the virus, it should be mentioned that they coincided with the presence of virus in this organ. In rabies and distemper, oxy- philic corpuscles, intra- or extra-cellular, staining red by Mann's method, have been described as concomitant with the presence of virus in the parotid. No actual corpuscles have been found by us in the parotid of animals infected with the virus of enzootic encephalo- myehtis. In the medullary zone of the adrenal small accumulations of lym- phocytic elements were occasionally seen. The lung, the liver, the spleen, the testicle, and the ovary appear macro- and microscopically normal. Characteristic lesions of an intense nature are found only in the nervous system. We may class these in two categories, (1) infiltrative and (2) degenerative. Both types of lesions may be met with in the brain, mesencephalon, cerebellum, spinal cord, and spinal ganglia. In the nerve-roots and peripheral nerves (sciatic and brachial nerves examined) only infiltrative lesions have been found. (2) Lesions iii the Central Nervous System. (a) The Brain. The pia mater is infiltrated with mononuclear leucocytes, varying in individual cases. In some areas only a trace of this infiltrative process may be seen, while in others three or four layers of infiltrative cells occur. They are especially marked in the region of the meningeal vessels as well as in the spaces between the convolutions, and may form actual meningeal plaques. The infiltrating elements are lymphocytes, plasma cells, and large mononuclears. Vessels of the pia mater are often surrounded by ' cuffs ' constituted by mononuclear leucocytes (Fig. 10). The most intense lesions of the meninges are generally found at the base of the brain. In sections 54 BOENA DISEASE AND cut at right angles to the surface of the brain vessels passing from the meninges into the cortical substance surrounded by lymphocytic 'cufling' have often the aspect of septa (Fig. 11). In the cortex diffuse infiltration of lymphocytes accompanied by proUferation and mobili- zation of the neurogUal cells occurs. Especially in the liippocampus (the so-called 'elective zone' in herpetic encephahtis of the rabbit), the neuron degenerates, the nucleus swells, the chromatin becomes rarefied, collects towards the periphery of the nucleus, and in its place appear small oxyphihc globules which may be at the hmit of visibihty or may reach the size of a nucleolus. Some of the neurogUal cells of this region appear to undergo the same degenerative process. It is similar to, although less intense than that described by Levaditi, Harvier, and Nicolau (1922) in experimental herpetic encephalitis in the rabbit, leading to the formation of encephalitic neuro- corpuscles, and is not of a specific nature. The oxyphilic degenera- tion of the nucleus may lead to the formation of larger corpuscles surrounded by a halo, morphologically identical with those described as specific in Borna disease by Joest and Degeu (1909). The nucleus may react in the same waj' to other causes. Even the halo is not wanting in the figures given by Levaditi, Harvier, and Nicolau. We regard this phenomenon as possibly the result of the action of a karyotropic virus, the degenerated karyoplasm fusing round the pathogenic agents These nuclear lesions may be found in all regions of the brain, although the German writers have described their presence only in the Cornu Ammonis, in which, indeed, they are more con- stantly found. They may be single or in pairs surrounded or not by a ciiaracteristic halo. When sections are stained with Mann's stain or toluidin blue-eosin, the intranuclear corpuscles are stained rose or red. while the nucleolus is more of a violet tint. They occur also in the pyramidal cells of the cerebral cortex and even in the neuroglial cells (large granulo-adipose cells of the hippocampus). In the Ammon's horn newly formed capillaries are sometimes seen. The vessels of this region appear dilated, gorged with l)lood, and their adventitia infiltrated with several layers of mononuclear leucocytes. Plasma cells are abundant in the process of perivascular infiltration. We have never found such a large number in rabies of the dog or rabbit, in pohomyehtis of monkeys, in human encephalitis, in chronic herpetic encephalitis of rabbits, or in the vascular lesions produced by the presence of the so-called ' Encephalitozoon cuniculi '. Small lymphocytes may also be found in large numbers in these perivascular infiltrations, but large mononuclears cells are also present to a less extent. The presence of polymorphonuclear leucocytes is excep- tional. Sometimes in the thickness of the 'cuffing' are degenerated lympho- cytes or plasma cells. The nucleus of these degenerated cells has become intensely oxyphilic. It is reduced in size and condensed in 'blocks' without any definite structure. When Mann's stain is used, the protojilasm, in the case of the degenerated plasma cells, stains rose; the unchanged elements stain bhir. Here and tliere in the ENZOOTIC ENCEPHALO-MYELITIS 55 mass of the infiltrating cells rare fragments of degenerated chromatin can be found, probably arising from degenerated mononuclear leucocytes or pyknosed polymorphonuclear cells. The intensity of the infiltration varies. Sometimes the perivascular 'cuffing' is con- stituted by one, two, or three layers, while in other cases a massive nodule resembling a gumma may he found with a small vessel in the centre. These alterations occur not only in the vessels of the Ammon's horn, but in all the regions of the central nervous system where 'cuffing' may be found. In the Cornu Ammonis areas of mononuclear infiltration are seen between the ganglion cells, in the row of fusiform cells, or in the chain of small granular cells (Fig. 12). Neuroghal cells of neoformation may also participate in this infiltrative process. We have never found true neuronophagia at this site, but we have encountered a curious phenomenon, the exact nature of which we iiad some trouble m determining. In a section of brain examined under the oil immersion we found in one of the large ganghon cells of this region stained with toluidin blue-eosin a species of ' cyst ' in the interior of the protoplasm (Figs. 13, 14. 15, 16). The rest of the cell preserved its normal aspect. The 'cyst' compressed the nucleus and pushed it towards the periphery of the cell, forming a marked depression in the nuclear membrane. The diameter of this ' cyst ' was about 7 IX. It was marked off from the cytoplasm of the neuron by a membrane, and contained six basiphilic granules of equal size, placed symmetrically at its periphery. On subsequent minute examination of the cells of the Ammon's horn we found these ' cysts ' again in several preparations. They have been found only in this region and always in the protoplasm of the cell. Their diameter varied from 5 /x to 9 /x. During the search we have seen occasionally plasma cells, the nucleus of which had undergone degeneration, which in its appearance recalled this intracellular 'cyst'; the karyoplasm was condensed into several small round in- tensely chromatophihc granules, apparently attached at equal dis- tances to the nuclear membrane. The presence of a protoplasmic circle around this formation removed from our minds the supposition that we were deahng with a ' micros poridian cyst '. and showed dis- tinctly that it was a degenerated infiltrating cell (PI. I, Fig. 3). The staining reactions indicated that the cystic formations were the degenerated nuclei of the plasma cells which had penetrated into the interior of the large cells of the Ammon's horn. We have found such bodies in approximately 5 per cent, of cases examined. They have never been met with in the brain of normal rabbits, or in the brain of rabbits which have succumbed to infection with the viruses of herpes, rabies, or vaccinia. We have described these formations in detail, since the elucidation of their nature required extended observations. Many control animals were examined to ensure that we were not dealing with a spontaneous ' microsporiduin ' disease of the rabbit. Pathological changes other than the above are found more con- 56 BOENA DISEASE AND stantlv in the Amnion's horn, especially in cases showing intense intiltrative lesions. For instance, a number of the nerve-cells may show degenerative changes characterized by the following appear- ances. The nucleus appears oedematous, the chromatin is fragmented, while the protoplasm shows more or less advanced tigrolysis. The protoplasm also contains vacuoles and the cellular membrane is denticulated. In some cells, also, of the Ammon's horn, the nucleus, and sometimes too the protoplasm, becomes oxyphiUc. The fusiform cells which are found in the upper part of the row of large ganghon cells show intense nuclear degeneration: the karyoplasm is con- densed in a 'block' and stains red with Mann"s stain. When a pre- paration so stained was decolorized gradually, and examined after each stage of the process until these degenerated nuclei liecame a pale rose colour, it was found that they had no definite structure, but consisted of a round mass of homogeneous condensed chromatin much smaller in size than the unchanged nuclei. This type of nuclear degeneration has also been met with in the mesencephalon (PI. Ill, Fig. 4). We have ahready referred above to the fact that oxyphilic corpuscles which may or may not be surrounded by halos are found in the nucleus of some of the cells of the Amnions horn. They can be distinguished from the nucleolus by their different staining reactions (PI. I, Fig. 2). These intranuclear corpuscles— the specific 'inclu- sions' of the German workers — may be single or in twos or threes, varying in size. Sometimes they may be at the hmits of visibility, or they may be as large as 2 /x or 3 fi. These corpuscles occur else- where. They may be found in the cytoplasm of the cell and are possibly expelled intra vitani. but as it is possible for the nucleolus of a cell to be dislodged by the microtome knife, the same factor might carry the intranuclear corpuscle into the cvtoplasm. Our opinion is that the intranuclear 'inclusion' in Horna disease is possibly a reaction of the karyoplasm against the pathological agent which penetrates the interior of the nucleus. Possibly the chromatin masses around the infective virus elements. This is suggested by the staining reactions, since the condensed mass of chromatin wliich forms the corpuscle undergoes degeneration from the centre towards the periphen,- (the centre appears oxyphilic and the periphery basi- philic in certain corpuscles). Around the lateral ventricles well-marked infiltrations are found, these being in some cases very intense. The choroid plexus is also infiltrated. The epitheUuni of the ventricle and of the ependyma is unchanged. Occasionally in the parenchyma in the region of tht! ventricle, or even in the cerebral cortex itself in the superficial areas, karyo- kinetic figures may be seen. Probably this karyokinesis is in niol)ilized cells of the vascular endothelium which have penetrated into the ner\'e substance. In certain preparations we have seen 2, 8, or even 4 karyokinetic figures. The mitosis sometimes undergoes oxyphilic degeneration. ENZOOTIC ENCEPHALO-MYELITIS 57 There are lesions affecting the area above and below the ependyma and also the surrounding zone: a mobihzation of the neurogUal elements takes place, while at the same time lymphocytic elements are found in the immediate proximity of the neuron. Up to a certain point this phenomenon is comparable with the 'satellitism' described by Metchnikofi' in senihty. Certain of the nerve-cells are surrounded on all sides by 'satelhte' cells and cells of infiltration, which occa- sionally penetrate the interior of the cell. Six, eight, and even ten neurons may be seen ' besieged ' in one microscopic field. The cells which come in immediate contact with the nerve-cell push in the cellular membrane and form ' cups ' in the periphery of the cytoplasm, giving the nerve-cell a denticulated border. This phenomenon is more com- monly met with in sections from rabbits dying within the first 20 days after inoculation. The intensity of 'satellitism'. is in inverse propor- tion to the meningeal and perivascular lesions. When the meningitis and perivascular cuffing are at a minimum ' satelhtism ' may represent the only departure from the normal discovered in the brain. From the examination of a large number of preparations we are led to beheve that 'satelhtism' is a stage which may either disappear during the evolution of the disease, resulting in a quasi-normal state, or become intensified and be followed by neuronophagia. We have found neuronophagia present in sections of brain showing marked 'satellitism' (Fig. 17). Our conception of the various stages of the struggle against the virus is as follows: When the nervous system is invaded by the virus the neuroghal elements and mono- nuclear lymphocytes are attracted to the parasitized neuron. If the neuron succeeds in freeing itself from the virus, the local reaction ceases at this stage, and the resorption of the satellite elements follows: but if the neuron dies in the struggle against the virus after undergoing intense degeneration, it is invaded by the satelhte cells and rare polymorphonuclear leucocytes ; the process has now reached the stage of neuronophagia. When the struggle between the neuron and the virus terminates without neuronophagia taking place the animal survives until the meningeal lesions, perivascular infiltrations, and infiltration of the Amnion's horn become incompatible with life. In this case death takes place at a later stage, i.e. in from 25 to 50 days. These conclusions as to the evolution of the morbid process in the brain, formed from observations on the character and position of the lesions in a large number of rabbits dying early or late after inocula- tion, have received further support from the study of the histogenesis of the alterations in the central nervous sytem of six rabbits killed at regular intervals after inoculation, i.e. on the 5th, 10th, 15th, 20th, and 27th day, and of one which died on the 31st day. In these animals also, the microscopical examination of sections of various parts of the nervous system showed that the first modification in the central nervous sytem is the mobihzation and prohferation of the neuroghal cells around the neuron — 'satellitism'. this being more marked in the pons and the medulla oblongata. In the process of 'satelhtism' one finds not onlv neuroglial cells but also mononuclear 58 BOENA DISEASE AND cells taking part. Later, infiltration of the Cornu Ammonis, the meninges, and the vascular tissues takes place. In the subjects of our experiments the latter process began to appear towards the 15th or •20th day after the inoculation. The presence of polymorphonuclear cells is quite exceptional no matter at what stage of the infection or from what site one examines sections of the central nervous system; the infiltrative lesions are constituted from the beginning by mononuclear cells. There are notable differences between the development of the encephaUtis of Boma disease in the rabbit and chronic herpetic encephaUtis produced experimentally in the same species. In the brain infected by herpes an acute stage is observed in which poly- morphonuclear leucocytes take part, and are fomid in large numbers in the perivascular 'cuffing' as well as in the nodular lesions at the base of the brain in the region of the hippocampus. If the animal survives this acute stage and recovers what have been termed by Levaditi and Xicolau (19'2'2) 'lesions d'imnmnite' may be found. These are small nodular or diffuse areas of parenchymatous infiltra- tion situated in the hippocampus (the 'zone elective') and are pro- duced by mononuclear cells that have replaced the polymorpho- nuclear leucocytes with which the inflammatory process commenced. In those rabbits which just fail to resist the disease and die in 20 to 30 days, the lesions are more intense, but, as in the case of the 'lesions d'immmiite', the infiltration consists of mononuclear cells which have taken the place of the polymorphonuclear leucocytes that predominated in the acute stage of the inflammatory process. In the case of the infection of the brain of the rabbit witli tiie virus of Boma disease, polymorphonuclear leucocytes do not plaj' a part in the early inflammatory process; during this early stage, one observes only 'sateliitism' of the neuron, while the infiltrative lesions are produced by mononuclear cells alone from the beginning of tiie process until the final stage. (b) The Mid-brain and Medulla Oblongata. In tlie mesencephalon and the medulla ol)l()ngata similar infiltrative and degenerative lesions occur. 'Cuffing' of tlie vessels is frequently observed. Certain of the nerve-cells appear to be in a state of advanced tigrolysis (Xissl's granules have disappeared). Degeneration both of tiie nucleus and the cytoplasm is frequently found. Certain of the cells appear to have their pnjtophism split up, the nucleus lieing jieripheral, swollen, and completely degenerated (Fl. I, tig. 1). Occasionally typical neuronophagia is encountered. Iti tiiese regions also we have demonstrated the presence of the intranuclear cor])uscies of Joest- Degen. The cells which contain them generally preserve otherwise their normal aspect ; the nuclear membrane is intact, wliile the proto- plasm is structurally unchanged and stains normally. The converse is also true; we liave never found the corpuscles of Joest-Degen in cellH in advanced stages of degeneration or disintegration. Negri liodies in rabies are also oidy found in nerve-ceils wiiicii are otherwise normal. In tlie mesencepliaion, as mentioned above, one meets most ENZOOTIC ENCEPHALO-MYELITIS 59 frequently with ' satellitism ' of the neuron, which in certain cases goes so far as to constitute true neuronophagia (Fig. 18). In the mesen- cephalon also we may tiud small islands of mononuclear cells in the parenchyma without any relation to the vessels. In certain cases we found neuroglial cells (granulo-adipose cells) showing nuclear oxy- chromasia and occasionally small oxyphihc corpuscles within the nucleus, similar to the so-called 'encephahtis neurocorpuscles ' of herpes described by Levaditi. Harvier, and Nicolau (1922). (c) Cerebellum. In the case described the lesions in the cerebellum were much more intense than the average, since usually the altera- tions consisted only of shght meningitis and perivascular infiltration accompanied by occasional 'satelhtism' of the cells of Purkinje. In the septum there was a marked infiltration with mononuclear cells. Massive perivascular 'cufiing' was present, especially in the white substance between the convolutions. In the vessels themselves, which were gorged with blood, an excessive number of mononuclear cells were found. There was an intense infiltration in the granular layer, which in some cases was completely destroyed and replaced by areas formed exclusively of mononuclear cells. Here and there in the islands of lymphocytes 'basket' cells -vsith a pale-staining degenerated protoplasm were found, their border appearing irregular. Several of the cells of Purkinje appeared to be hyperchromatic, and their nucleus was in some cases eccentric and stained by acid stains. In others the nucleus was not separated from the rest of the proto- plasm since the remains of the nuclear membrane appeared to have disappeared. Other Purkmje cells were degenerated and appeared as cell shadows. In other parts the karyoplasm of certain of these cells was condensed around the nucleolus. Satellitism of the basket cells and the cells of Purkinje was noticeable, but true neuronophagia was not seen. The lesions were confined to certain areas; other parts of the cerebellum were perfectly normal. In a case where the inoculation of the virus was made by the intratesticular route the lesions in the cord were especially well marked, and in the cerebellum the satelhtism of the cells of Purkinje was occasionally so advanced as to constitute almost a true neurono- phagia. (d) The Spinal Cord. Generally it may be stated that the intensity of the lesions found in the brain or in the spinal cord corresponded with the intensity of the sj^mptoms observed during life. In the rabbits we have examined pathological changes were always present in the cord whether symptoms of affection of this part of the central nervous system were present or not, but these were much more intense when the symptoms produced by affection of that region domuiated the cerebral symptoms. As in the case of the brain, the meninges of the cord are not as a rule greatly affected. Only in isolated cases was a severe meningitis found. The anterior and posterior septa may be more or less infiltrated with mononuclear cells. Perivascular 'cufiing' is seen both in the 60 BORNA DISEASE AND grey and white matter of the cord. In the anterior and posterior horns infiltrations with mononuclear cells may be seen. The most intense infiltrations are found in the posterior horn, while in the anterior horn degenerative lesions of the neuron are more common. The process of degeneration in the nerve-cells is the same as that in other regions of the central nervous system: tigrolysis occurs, nuclear oxychromasia exists, while the whole cell shows a marked hyperchromasia. Vacuolization of the cytoj)lasm, a degenera- tive process, may also be seen (PI. II, Fig. 1), while in rare cases when the lesions as a whole have been exceptionally intense, occasional neuronophagia was recorded (Fig. 19). The phenomenon of 'satel- litism' appears to be more commonly met with in the cord than in the brain itself. The intranuclear corpuscles of Joest-Degen found in the cord have generally been in the nerve-cells of the anterior horn (PL I. Fig. 5; PI. Ill, Fig. 2). Here and there small islands of lymphocytes may be found in- filtrating both the white and the grey suiistauce, these islands being unconnected with vessels. Frequently it has been observed that the zone of Lissauer is the site of a well-marked mononuclear infiltration. The lesions found in the spinal cord are comparable with those found in poliomyehtis. The neuronophagia so characteristic in the cord of monkeys infected with the virus of the latter disease (to which rabbits are generally considered not to be susceptible) is also present in the cord of rabbits infected with the vn-us of Borna disease, but to a less degree. (3) Lesions in tite Peripheral Nervous System. (a) The Posterior Nerve-roots arising from the cells in the zone of Lissauer have infiltrative lesions which vary in intensity. This zone, as has i)een stated above, is generally infiltrated with mononuclear cells. The infiltration takes place i)etween tlie nerve filaments, and consists of a chain of lymphocytes. In some cases only traces of this infiltration can be seen, while in other cases massive perivascular infiltration may occur (Fig. 20). The interstitial infiltration in the anterior nerve-roots is very discrete or absent. This process of radiculitis has not been mentioned by other workers who have studied the disease in animals infected experimentally; nor lias the process of infiltration of the nerve-roots been described in the spontaneous disease. (b) The Spinal (Uinqlia. The most intense lesions in the peripheral nervous system have been found constantly in the spinal ganglia. The process of intiltration in the posterior nerve-roots becomes more intense as they enter the ganglion, and Ix'tweeii the nerve-fibres which ])!iss through the substance of the ganglion a well-marked monomiclear infiltration is seen. In the rest of the ganglion tbe lesions as a rule are very intense. The alterations in the ganglion and tbe various elements taking part in the infiltrative and degenerative i)rocesses at this site are always the same, no matter rn»ni wbiit individual case or from what region ENZOOTIC ENCEPHALO-MYELITIS 61 of the cord (cervical, thoracic, or lumbar) the ganglion is taken (Figs. 21, 22). The changes are as great and the lesions of the same importance when the ganghon originates from a case showing altera- tions in the cord which are scarcely discernible as from a case where such changes are very pronounced. The capsule of the ganglion shows neither infiltration nor degenera- tion. In the interior of the ganglion mononuclear interstitial infiltra- tion is abundant. The small intraganghonic vessels show perivascular 'cuffing". The infiltrating mononuclear elements are found dis- seminated between the nerve-cells or massed together forming actual nodules comparable with those described by Van Gehuchten and Nehs (1900) in rabies (Fig. 23). The mononuclear cells may be grouped together in small islands between the nerve fascicuh which traverse the gangUon. The ganghon cells themselves appear to be 'choked' by the infiltrative process in some microscopic fields. In certain parts these cells undergo profound changes: the nucleus becomes oxj'phiHc, the protoplasm loses its granular nature, assuming a homogeneous appearance, and becomes shghtly oxyphihc when stained with toluidin blue and eosin. We have found that the changes in the cells of the ganglion are more marked than in any other region of the nervous system; and the intranuclear corpuscles are larger and in greater number here than in any other site. In some microscopic fields the nucleus of every cell may contain one or two corpuscles of Joest-Degen surrounded by a halo. The most important and frequent type of lesion in the ganghon, however, is neuronophagia. Lymphocytes, plasma cells, and large mononuclears penetrate the peripheral zone of the neuron. One often finds a clear zone in the protoplasm around these infiltrating cells suggestive of the action of a proteolytic ferment hberated by the invading cells. Later the mass of detritus of the neuron is removed by the macrophages aided by occasional polymorphonuclearleu- cocytes. The number of infiltrating cells increases, the whole body of the nerve-cell being invaded, and finally, in place of the neuron, one finds nothing but a nodule formed by mononuclear cells (Figs. 23, 24, 25, and 26). The most intense lesions of both an infiltrative and degenerative character are found in the peripheral zone of the ganghon; this point will be discussed again later. (c) The Peripheral Nerves. In the peripheral nerves infiltrative lesions are also fomid. A detailed study has been made of lesions found in the sciatic and brachial nerves. The technique employed in carry- ing out this research was as follows : All the rabbits of which the sciatic and brachial nerves were sec- tioned for histological examination had been inoculated intracere- brally with the virus of Borna disease. We removed the terminal part of the cord (sacral) with the roots of the sciatic nerve and their various gangha together with a portion of the peripheral parts of the nerves. This whole was fixed in Duboscq-Brasil-Bouin fluid. Longi- tudinal sections were made after the manner figured (Fig. No. 27). We have found lesions in all cases examined, these being more 62 BOEXA DISEASE AND intense towards the origin of the nerve and becoming less intense towards its termination. The alterations consist of interstitial or peri- vascular intiltrations with mononuclear cells. The nerve-sheath is. as a rule, unaffected. In certain cases the infiltrations appear to 'dissect' the nerve filaments (Fig. 28). The whole process constitutes a descending neuritis produced by the virus propagatuig centri- fugally.^ Kecently G. Marinesco and S. Draganesco (1927) pubUshed their observations on the pathogenic process in herpes zoster. A complete chnical report is given of cases in wliich the locahzation of the lesions in the nervous system suggested to the authors that the infection commenced by an ascending neuritis followed by a gangho-radicuUtis and myelitis. 'Wohlwill (1924). Levaditi (1926), "Pette (1924), Foerster (1924), and others advanced similar hypotheses as to the centripetal propagation of the infection. In support of their theory as to virus ascending from the peripheral nerves. Marinesco and Draganesco refer to the lesions in the corresponding gangha: ' In the gangUon the most intense lesions were in the peripheral zone . . . this topography of the inflammation explains the spread of the infection hv the peri- capsular lymph vessels, to the interior of the ganghon." However, from a comparison of the description of the lesions pro- duced by the downward extension of the virus in rabbits infected with the virus of Borna disease, and those in herpes zoster, it will be seen that the histological pictures are identical. We have found lesions in the peripheral nerve not only close to the ganglion as de- scribed by Marinesco in zoster, but also in the terminal filaments farthest removed from the ganglion, showing that the virus in our experiments diffused by centrifugal propagation. These facts allow us to assume that the topography of the lesions is not a criterion by which to judge the portal of entry of the virus with a suflicient degree of accuracy. Comparable lesions can be produced in the central and peripheral nervous system both by infection intracerebrally or by inoculation of the virus into the sciatic, i.e. no matter whether the infection is ascending or descending. This example of lesions being produced in the peripheral nervous system after introduction of the virus into the central nervous system (brain), suggests the possibiUty of infection being central in origin in the case also of herpes zoster. \ similar pathogenic process is not excluded in recurrent herpes, peripheral forms of ei)idemic encepiia- iitis, and perhaps also in certain cases of sciatica. (4) Sumnuiry and Discussion. The inoculation of the virus of enzootic encephalo-niyelitis intra- cerebrally into rabbits produces changes in the nervous system, which are those of a meningo-encephalo-myelitis, a ganglio-radiculitis. and a peripheral interstitial neuritis. The lesions in the central nervous system as well as in the spinal ■ We h»ve ticf-n able to demonittratc tlio prcsoiirc rvc« by inuculatidii of their emuliiionii into the hrnin of ralibit« (Reo ]i. 40). ENZOOTIC ENCEPHALO-MYELITIS 63 ganglia are both infiltrative and degenerative. The meningitis and the perivascular and parenchymatous infiltrations are produced by mono- nuclear cells. Pathological 'satellitism' of the neuron is most pronounced in the mesencephalon, medulla oblongata, and spinal ganglia, but may be found also in other regions of the cord and brain. It may in some cases be so advanced as to constitute true neuronophagia. The latter phenomenon is most common in the paravertebral ganglia. The intranuclear corpuscles of Joest-Degen, considered by the present writers to be evidence of an attempt at defence by the nerve- cell, and referred to as specific ' inclusions ' by other workers, may be found in the various regions of the brain, cord, and spinal gangha. They are almost constantly present in the large ganglion cells of the Cornu Anmionis and the nerve-cells in the spinal ganglia. In our opinion the cell which reacts against the presence of the virus by the formation of intranuclear corpuscles has formed a barrier to the extension of the destructive action of the virus in 'blocking' the infective elements within a condensation of its chromatin. It is feasible to conceive that this process removes the virus and renders it inoffensive: for this reason the cell maintains its integrity. In those cases where the cell becomes degenerated or neuronophagia takes place, one may suppose that the nucleus has been incapable of sur- rounding the infective particles by condensation of its chromatin and thus limiting the extension of the activity of the virus. This failure to form intranuclear corpuscles may be due to the quality of the virus (virulence), the quantity of the virus, or the deficiency in the normal resisting power of the neuron, the result being that the virus multiphes and ultimately destroys the cell. One must recall that the figure described on p. 56 supports this view as to the method of production of the corpuscles of Joest-Degen. The infiltrations in the nerves are interstitial in character and are produced by the invading mononuclear cells arranging themselves in chains between the nerve filaments. Perivascular ' cuffing' also occurs. A peripheral interstitial neuritis occurs in Borna disease after the introduction of the virus into the brain, and the authors, without excluding the possibility of ascending infections, have suggested that herpes zoster, ^ recurrent herpes, the peripheral forms of epidemic encephalitis, and perhaps also certain forms of sciatica may be the secondary manifestations of a disease, the original focus of which is in the central nervous system. The hypothesis has already been advanced (see p. 88) that in cases of infections with these viruses central infection of the brain takes place; but the central nervous system, being able to resist the action of the virus more efficaciously, shows no manifest disturbances, while the peripheral nerves, poor in methods of defence, do not rid them- selves of the infecting elements which proUferate and produce lesions. ' The work of Head and Campbell (1900) on the pathology of herpes zoster also suggests that zona is a secondary peripheral manifestation of a disease originating in the central nervous system. 64 BOENA DISEASE AND B. The Guinea-pig. Macroscopic Examination. The brain and spinal cord appeared congested. No other organ showed pathological changes except the stomach, in which occasionally the non-specific lenticular haemor- rhagic areas, similar to those described in the rabbit, were found. Microscopic Examination. Lesions were fomiil in the central and peripheral nervous sytem and in the kidney. The alterations in the stomach wall, when they existed, were comparable with those found in the rabbit, namely, autodigestion of the mucosa with small loeahzed haemorrhage. As in the case of the rabbit, the kidneys showed marked congestion, but no actual nephiitis was recorded. Central Nerrous System. The lesions found in the brain were similar to those in the rabbit, except that their intensity was less. The intra- nuclear corpuscles of Joest-Degen were usually found in the Cornu Ammonis and elsewhere (PI. I, i"ig. 4). Infiltrative and degenerative lesions characteristic of the disease were found (PI. Ill, Fig. 8). The lesions in the mesencephalon, cerebellum, and spinal cord were as in the rabbit. The intranuclear corpuscles of Joest-Degen were frequenth' present in the anterior horn of the spinal cord. Peripheral Nervous System. Infiltrative processes with mono- imclear elements were found in the nerve-roots, but they were not so marked as in the rabbit. The lesions in the spinal ganglia were similar in nature to those described in the rabbit, but were not so acut<». The lesions in the peripheral nerves were more intense in that portion nearest the ganglia. C. The Rat and Mouse. The organs, except the nervous system and the kidney, were macroscopically and microscopically normal. In the brain and spinal cord the lesions found were similar to those in the rabbit and the guinea-pig. The intranuclear corpuscles of Joest-Degen were found in the brain of rats and mice. Infiltrative lesions of a discrete nature were also found in the sciatic nerve of the rat. D. The Monkey (Macacus rhesus). Our description of the lesions found in the monkey is made from a study of sections from different parts of the nervous system of Monkey M. 1 {Macaciui rhrsu.<>). The protocol of the experiment in which this animal was infected is given on p. 84. Similar lesions were found in Monkey M. 8. although the clinical pictun* in the case of the latter monkey was diffrrent from that of M. 1. The macroscopical and microscopical examination of the spleen, liver, pancreas, lung, myocardiuui, testicle, ovary, parotid gland, and the mesenteric and inguinal lymph glands did not reveal any patho- logical changes in these organs; the kidneys were iiyperaemic. In the adrenal gland there was slight infiltration with lymphocytes in the medullary zone, the lymphocytes being disseminated in tlie ENZOOTIC ENCEPHALO-MYELITIS 65 parenchyma or grouped together in small islands. The brain appeared to be normal by naked-eye examination. (1) Lesions in the Central Nervous System. (a) The Brain. Frontal Lobe. Meningitis of a mild character was present in some areas, becoming intensified near those vessels in which slight perivascular infiltrations were observed. The patho- logical process in the meninges might be described as an 'irritation' rather than a true meningitis, while the perivascular 'cuffing' con- sisted of three or four layers of cells only in the walls of the vessels in contact with the brain ; in the rest of the vessels the process of infiltra- tion hardly existed. Certain of the small vessels penetrating the cerebral parenchyma from the meninges were surrounded by charac- teristic ' cuffing '. There was infiltration with mononuclear cells in the septum. The lesions in the meninges, the 'cuffing' (Fig. 30), and the infiltration of the septum, were produced by lymphocytes, plasma cells, and macrophages exclusively. In the parenchyma, and especially in the white matter, extensive 'cuffing' of the vessels could be seen, consisting of ten to twenty layers of infiltrative cells. Some of the pyramidal cells appeared to be degenerated. Intense satelhtism of the neuron was present in some instances, and in certain of the cells oxyphilic corpuscles surrounded by a halo of the type described by Joest and Degen were found (PI. Ill, Fig. 1). The karyoplasm was rarefied in the greater number of the cells containing 'inclusions', suggesting that the degenerated chromatin was condensed in the corpuscles. In the deeper part of the brain, both satelhtism and the intranuclear corpuscles of Joest- Degen were less frequent than in the peripheral zone. Parietal Lobe. The meninges were infiltrated with mononuclear cells (Fig. 29) which formed plaques in certain regions. There was discrete infiltration of the septum. In the brain substance, peri- vascular infiltrations, consisting of many layers of cells, were found forming small nodules: that these were perivascular was evidenced by the presence of a small vessel in the centre of the nodule (Fig. 31). No neuronophagia was recorded in this region of the brain, although the acute ' satelhtism ' of the neuron sometimes suggested the pheno- menon. Occasional nerve-cells in a state of degeneration had eccentric nuclei and their protoplasm was undergoing tigrolysis. No actual parenchymatous infiltration could be seen, but rare mononuclear cells were dispersed in the parenchyma. A large number of nerve- cells in this region contained large oxyphihc corpuscles surrounded by halos within the nuclei. Occipital Lobe. Meningitis was rarely observed in this region of the brain. Certain of the meningeal vessels had several layers of mono- nuclear cells on their walls in contact with the brain. In the septum, infiltration was not well marked, although several venules were sur- rounded by 'cuffing'. Eich perivascular infiltrations were found in the parenchyma, more especially near the large pyramidal cells. Pathological 'satelhtism' of the neuron by neuroghal elements and 66 BOEXA DISEASE AND occasional lymphocA'tes was not uncommon in the occipital lobe: these satelhte cells sometimes produced marked depressions in the protoplasm of the host cell. The intranuclear corpuscles were of very much smaller dimensions and were observed less frequently than in the parietal lobe. Hippocampus. The lesions in the meninges were similar to those found in the occipital lobe. The perivascular 'cuffing' in the paren- chyma was poor in elements. There was a slight infiltration with mononuclear cells between the large and small pyramidal cells. The number of 'inclusions' was greater in this region than in the occi- pital lobe. Cornu Ammonis. In this region of the brain the ' cuffing ' around the vessels was so extensive as to suggest a nodule or pseudo-gumma : the presence of a small vessel in the centre of the nodule was discerned with difficulty. In addition, there were small groups of mononuclear cells bearing no relation to vessels. In certain cases 'satelhtism' was so advanced that it could almost be described as neuronophagia. The large ganghon cells preserved their normal structxire and nearly all contained within their nucleus well-marked corpuscles of the Joest- Degen type. Basal Ganglia. Perivascular ' cuffing ' was observed. In some of the degenerated nerve-cells the nucleus could not be differentiated from the rest of the protoplasm. Pathological 'satellitism' of the nerve- cells was intense. Intranuclear corpuscles were found on occasion, not only in the nucleus but even in the cytoplasm. Small islets of mononuclear cells were also found. Pons. The process of perivascular infiltration was abundantly present in tliis region. Many of the ner\e-cells were in an advanced stage of degeneration, while the nuclei were swollen and unrecognizable as such. The proto- plasm of the cells was in a state of tigrolysis and its contour was broken in many places by splitting. The cells containing intranuclear corpuscles were othenvise morphologically normal. No areas of infiltration were seen. (b) Cerebellum. There was slight infiltration of the meninges and septum with mononuclear cells: tiie parenchyma was not infiltrated. The layer of the small granular cells had a normal aspect. CiTtain of the cells of Purkinje were degenerated. In some of the latter cells oxyphihc corpuscles with a characteristic halo were recorded. (c) Medulla Oblongata. Meningeal changes were slight. Vessels were normal without 'cuffing' and the parenchyma was not infiltrated. Occasional neurons showed evidence of commencing degeneration, and in certain of these large oxyphilic corjjuscles were present. (dj Spinal Cord. Cervical liegion. I'he meninges were normal and no 'cuffing' was present in them or in the grey or white matter. A slight diffuse inliltration was ol)served. In the anterior horns non- degenerated neurons had intranuclear corpuscles (Joest-Degen type). In certain regions satellitism was a marked feature. The zone of the cells of Lissauer was infiltrated with lympliocytes. The posterior ENZOOTIC EXCEPHALO-ifYELITIS 67 tiCTTe-roofe showed interstitial infiltration and sometimes even 'cuffing' in the vessels. The corresponding s-pinal ganglia showed intense inter- stitial infiltrations: only rare polymorphonuclear leucocytes were found. The protoplasm of certain of the cells in the ganglia had undergone tigrolysis, and in some cases also had become oxyphiKc; the nucleus of these affected cells was eccentric. Neuronophagia was frequently seen and nodules of mononuclear cells comparable with those found in rabies were not uncommon. Large intranuclear corpuscles were found in nerve-cells of the gangUa which otherwise preserved their morphological integrity (Fig. 35). Thoracic Begion. No meningitis was present, nor was there any evidence of 'cuffing'. There was sUght infiltration of the posterior horns and a concomitant degeneration of certain nerve-cells of the anterior horns. In the latter region also rare intranuclear corpuscles were found (PI. II, Fig. 2). The posterior nerve-roots showed inter- stitial infiltration and ' cuffing ' around the vessels. Lumbar Region. No perivascular infiltrations and no meningitis could be seen. There was a slight diffuse parenchymatous infiltration with lymphocytes. Satelhtism of the neuron was present, both in the anterior and the posterior horns. There were well-marked lesions in the lateral honis: the protoplasm was markedly oxyphilic and the nuclei of the cells were eccentric. Sometimes the sateUite cells pene- trated the protoplasm of the degenerated neurons and constituted almost true neuronophagia. Certain small nerve-cells of the anterior bom showed neuronophagia. Intranuclear corpuscles were rare. Inter- stitial infiltrations and 'cuffings' were seen in the posterior nerve- roots (Fig. 34). In the spinal gangha the lesions were of a similar nature to those encountered in the upper regions of the cord, but were more intense (Fig. 86). Neuronophagia was a constant feature. The nuclei of cells containing intranuclear corpuscles of the Joest-Degen type showed a rarefication of the karyoplasm, but no other morphological changes were present in such cells (PL III, Fig. 5). Sacro-caudal Region. In the large nerv^e fasciculi rich infiltration with mononuclear cells could be seen, together with marked 'cuffing' round the vessels (Fig. 33). In the posterior roots of the ner^^es the interstitial infiltration and 'cuffing' round the vessels was rich. The spinal gangha showed similar lesions to those recorded in the lumbar region. The capsules of such gangha were unaffected, and the lesions were marked in the periphery of the gangUon. In the nerves, after their exit from the ganghon, lesions similar to those seen in the nerve-roots were found. Massive infiltration with mononuclear cells was seen between the fascicuh of the nerves (epmeurium), in the connective tissue around the nerve (perineurium), and even in the endoneurium. ' Cuffing' of the vessels was also seen. (e) Sciatic Nerve. A study of transverse sections from the sciatic ner\"e after its exit from the greater sciatic foramen showed that there was a diffuse infiltration between the ner\'e fascicuh; this was E 2 68 BORNA DISEASE AND generally more pronounced in the interior of the fascicuh. In the thickness of the nerve an intense perivascular infiltration was seen (Figs. 38 and 39). Often the connective tissue of the sheath was unchanged, while intense infiltrative lesions were seen in the thick- ness of the nerve. Examination of longitudinal sections made from the sciatic nerve half-way between the greater sciatic foramen and the pophteal region showed that interstitial infiltration with mononuclears existed along with massive perivascular 'cuffing', while the nerve-sheath itself appeared perfectly normal. (f) Brachial Nerve. The alterations seen in tliis nerve were analogous to those described in the sciatic nerve; even in the lower third of the fore limb intense lesions were found (Fig. 37). The infiltrative lesions of the nerve-roots and peripheral parts of the nerves, as well as the alterations in the paravertebral ganglia, show that in the monkey, as in the rabbit infected intracerebrally with the virus, the infective agent travels from the central nervous system to the periphery along the nerves, producing the lesions described above, which constitute a ganglio-radicuhtis and descendiny peripheral inter' stitial neuritis. (2) Summary. In the brain there existed a mild meningitis with perivascular infiltrations and diffuse parenchymatous infiltrations, which some- times formed actual nodules; massive perivascular infiltration was also seen in the parenchyma; there was degeneration of nerve-cells and satelhtism. Intranuclear corpuscles of the type described by Joest and Degen were more easily found and more numerous than in the rabbit. In the cerebellwn there was found a slight meningeal reaction with perivascular 'cuffing', satelhtism of the cells of Purkinje in certain areas, and rare oxyphilic corpuscles (Joest-Degen type) in the nuclei were recorded. In the spinal cord no meningitis was recorded and there was an absence of 'cuffing' in the vessels of the coverings of the cord. In the posterior horn, a diffuse infiltration was observed, this being more marked in the lumbar region. In this region of the cord also a similar infiltration was seen in the anterior and lateral horns. In the anterior horns degeneration in the nerve-cells was recorded, and sometimes ncuronophagia. In the peripheral werroMS system an infiltrative radiculatis existed. The spinal ganglia were intensely affected, showing lesions both infiltrative and di'generative; neuronophagia was a connnon feature. A peripheral neuritis {sciatic and lirachial nerves) was present, con- sisting of interstitial and jicrivascular infiltration. The topography of the lesions as a whole shows that the virus intro- duced into the brain produces lesions locally, spreads to the rest of the central nervous system, and finally travels down the peripheral nerves. The cells in tlie lesions are almost exclusively nionoiiuclears. ENZOOTIC ENCEPH.\LO-MYELITIS 69 11. IMMUNITY All authors agree that in the horse an attack of enzootic encephalo- myehtis contracted spontaneously does not render this animal im- mune to a second attack; with regard to the disease in cattle and sheep, no precise records are available as to this point. The rabbit appears to behave differently. Zwick (1926) and his collaborators obser%'ed that in one case a rabbit which had been infected experi- mentally and had shown symptoms typical of the disease ultimately recovered and resisted a second intracerebral inoculation. The same authors succeeded, although not constantly, in producing a solid immunity by repeated inoculation of virus either subcutaneously or intravenously. They showed hkewise that the introduction of a large quantity of virus intraperitoneally may render rabbits refractory to subsequent infection. Ernst and Hahn (1926) have shown that the inoculation of virus intracerebrally into rabbits does not lead to the development of the disease, if, during the period of incubation, such animals receive repeated inoculations of virulent material into the veins. Zwick and his collaborators (1926), in the few experiments which they record in attempts to demonstrate antibodies in the serum of immunized animals by neutraUzation of virus in vitro, did not obtain very conclusive results. In passaging the strains of virus with which we have been working, viz. a strain originating from horses (Zmck) and a strain originating from sheep (Miessner), we have never found any healthy iminoculated rabbit refractory to infection. I. Attempts at Conferring Immunity to Rabbits by Inoculations of Non- attenuated Virus. A. Intravenous Inoculatian. (1) Four rabbits were inoculated intravenously with 1-5 corns, of a virulent centrifugahzed emulsion, which, M^hen inoculated into the brain of a rabbit, produced the disease and death at the end of 39 days. The rabbits inoculated intravenously did not show any symptoms and gained weight. Between 91 and 206 days later they were inoculated intracerebrally with fresh passage virus. Table X. below shows that a single intravenous inoculation of virulent emul- sion does not produce immunity in rabbits. (2) Another rabbit. No. 69, weight 1,980 gms., received three inocu- lations of virulent material into the veins. 16.5.27. First intravenous inoculation of 3 c.cms. of centrifugalized emulsion. 21.5.27. Animal normal. Weight 1,780 gms. 29.5.27. Animal normal. Weight 1,640 gms. 30.5.27. Second intravenous inoculation of 5 c.cms. of virulent emulsion. 8.6.27. Animal normal. Weight 1,600 gms. 13.6.27. Third intravenous inoculation of 1-5 c.cms. of virulent emulsion. 14.6.27. Animal normal. Weight 1,680 gms. 22.6.27. Animal normal. Weight 1,670 gms. 70 BOEXA DISEASE AND X ¥i « C5 ^ ES « « « G Q Q S § 8 g lO ■* OS 00 N e< C< IN :g"3 „ ^. ^ ttt " • ■ S S N S *-■ o 00 a> " M *^ '^ ENZOOTIC ENCEPHALO-MYELITIS 71 On 23.6.27 the rabbit received a test inoculation of virulent material at the same time as a control. The control died after 34 days with symptoms and lesions characteristic of the disease in the central nervous system. The rabbit prepared by intravenous inoculations behaved as recorded below. 3f0.6.27. Animal normal. Weight 1,7.50 gms. 7.7.27. Animal normal. Weight 1,950 gms. 10.7.27. Animal normal. Weight 1,680 gms. 14.7.27. Commencement of the disease. Weight 1,580 gms. 23.7.27. Typical symptoms of the disease. Weight 1,400 gms. ■• 26.7.27. Animal died during the day. Weight 1,230 gms. Microscopic examination of the brain and spinal cord revealed the presence of characteristic lesions. In this case three intravenous inoculations did not lead to immunity to cerebral infection. That four intravenous injections of virulent material may, however, produce a soUd immunity is shown by the experiment recorded on p. 75, Babbit 67. That the intracerebral inoculation of a filtrate (Handler filter) of virulent emulsion does not lead to the development of immunity may be concluded from the experiments mentioned on p. 13. The effect of introducing a thick emulsion of virulent brain into the trachea of rabbits was tested, but it did not produce the disease nor immunity in animals so treated. 0-5 c.cm. of a virulent emulsion of brain was introduced mto the trachea of three animals. As they did not present any morbid symptoms for 174 days they were inoculated intracerebrally with a virulent emulsion. They all died from enzootic encephalo-myehtis after 37, 27, and 40 days respectively. (See p. 33.) Eabbits which survive corneal or intratesticular inoculations of virulent material may become refractory to the disease as tested sub- sequently by intracerebral inoculation. B. Corneal Inoculation. The experiment recorded on p. 31 (Babbit 81s) shows that infec- tion by the corneal route can iminunize the rabbit against a sub- sequent inoculation by the cerebral route. C. Intratesticular Inoculation. Some measure of immunity may also follow intratesticular inocu- lation. Two rabbits (273 and 289) were inoculated into the right testicle with 1 c.cm. of a virulent cerebral emulsion. One of these rabbits, No. 289, died after 105 days. Typical lesions were found in sections of the various parts of the central nervous system, and pas- sage of its brain to a fresh rabbit gave a positive result. The other rabbit, No. 273, survived, and when inoculated by the 72 BOENA DISEASE AND intracerebral route 112 days later it proved to be immune. The control rabbit inoculated by the intracerebral route died on the 48th day. II. Attempts at Conferring Immunity to Rabbits by Inoculation of Attenuated Virus.^ A. Virus Inactivated by Ether. Roux ^ showed the attenuating action of ether on the virus of rabies. Later Eemlinger (1919) used an ether-treated virus sub- cutaneously to produce an immunity in rabbits to intracerebral inoculation ^vith fixed virus. Alvisatos (1922) and Hempt (1925) used ether-treated virus as a means of vaccinating man against rabies. Marie and Mutermilch (1927) have shown that one can immmiize rabbits against rabies by inoculating intrathocally virus treated with ether. We have tried to immunize rabbits against the virus of ence- phalo-myeUtis, employing a similar technique, with the difference that the virus treated with ether was inoculated into the brain; the proof of the avirulence of the virus treated with ether and the technique employed has been described on p. 22. The results obtained are given below. Two rabbits, 220a and 217a, weighing 2,450 and 1,950 gms. respectively, received two intracerebral inoculations of 0-5 c.cm. of ether-treated vims at an interval of 5 days. Twenty-one daj'S after the last inoculation they were inoculated intracerebrally along with a control rabbit, 14a, with fresh virus. Rabbit 217a died on the 42nd day, Rabbit 220a on the 17th day. and control Rabbit 14a on the 30th day after inoculation, and typical microscopic lesions were found in the central nervous system of all three. Thus two successive intracerebral inoculations into rabbits of virus treated with ether did not produce immunity to subsequent intracerebral inoculation. B, Virus killed by Chloroform. The proof that virus treated with chloroform is inactivated has already been given on p. 21. In the experiment about to be described, rabbits were twice inoculated intracerebrally with virus treated with chloroform, the interval between the inoculations being 5 days. The animals were tested 3 weeks after the second inoculation, and since the experiments were made at the same time as the experiments with ether-treated virus, the control Rabbit 14a served for both. The rabbits prepared with virus treated with chloroform succumbed ' The exporimrnU in attcmpU to immunize with virus trontod with ether, chloro- form, or formalin recorded are preliminary and for orientation ; it is possible that modificationH in technique might change the results originally obtained. There are at present under experiment other series of animals, and the retiults obtained in these ■ulwrquent attempts will lie recorded at some future date. ' Koux's unpublished olwervations. ENZOOTIC ENCEPHALO-MYELITIS 73 to a subsequent intracerebral infection after 37 and 42 days respec- tively. Cultures made from these brains were negative, but lesions characteristic of infection with Borna disease were found on micro- scopic examination of sections of brain, cord, and spinal ganglia. C. Virus treated with Formol. On p. 22 it was shown that virus treated with 0-2 per cent, formalin for 1 8 hours at room temperature failed to infect rabbits by the intra- cerebral route. The vaccine was prepared the day before use by subjecting fresh virulent brain material to the action of formahn in this concentration. A batch of ten rabbits was prepared by subcutaneous inoculation of this vaccine: the results of the experiment are recorded in Table XI. The results obtained in this experiment were not very satisfactory. Of the ten rabbits prepared by inoculation of formolized virus two died accidentally, five died following upon infection with the test dose given intracerebrally, and three proved to be immune to the test dose given intracerebrally. D. Virus Inactivated by Ultra-violet Light. An experiment on this subject is recorded on p. 20 in which a rabbit which survived after having received an inoculation of virus exposed for 5 minutes to the action of rays emitted by the mercury arc proved to be still susceptible to infection by the intracerebral inoculation of virulent material. III. Search foe Virucidal Antibodies in the Serum of Immunized Animals. The serum of the immune rabbit, 223a (see p. 75), was taken and mixed with equal parts of a centrifugahzed virulent cerebral emul- sion; at the same time two further mixtures of the same cerebral emulsion were made, one with equal parts of normal rabbit serum and the other with physiological sahne. The three mixtures were kept for 2 hours at 37° C. and were subsequently inoculated intra- cerebrally into rabbits. The results were: (1) the rabbit inoculated with the mixture of virus and immune serum died on the 14th day after inoculation; its brain passaged to a fresh rabbit killed in 35 days; the lesions found in sections of the brain of both of these rabbits were those of experimental Borna disease. (2) The control rabbits inoculated with the mixture of normal rabbit serum and virus died 39 and 34 days respectively after inoculation of Borna Disease, the lesions found on microscopical examination of sections of the nervous system being typical. (3) The rabbits inoculated with the mixture of virus and physio- logical sahne also succumbed to the inoculation. 74 BOENA DISEASE AND 5 -5 «i i « .Si N , -a C . i > § rt > « -S »^ ■£ S g « 1 " 5 < ■o '? -a -a '> -p ?Jt3 >^-3 g "3 fe "o o " Si»(55:cQ §1 I.: 5 « S «< ^ Si o c " § ^oooooo © coo .2:5iccc— "»sio o ooocc X 5>£ -OOOOOO O Sx^ ^ MOOOOOO « ^'^^ =< = ft >- 5 °5 s ii5l c^ t- CO :s M ^ ^ -^ < < < < < lO « 1^ 00 o> o ^ e^ C4 C« C4 94 ENZOOTIC ENCEPHALO-MYELITIS 75 IV. Experiments on Cross Immunity between the Strain OF Equine and that of Ovine Origin. Our experiments have been carried out with two strains of enzootic encephalo-myehtis, the one originating from the horse (Zwick's strain), and the other from the sheep (Miessner's strain). Experiment 1. Rabbit 223a. Weight 2,280 gms. A diluted emulsion of virulent brain (Zwick strain) which had been pulped and preserved in glycerine at room temperature for several weeks was inoculated into the brain on 22.10.26. The animal lost weight slightly, developed paresis of the hind quarters about the 20th day after the infection, but subsequently recovered, and on 15.12.26, 54 days after the inocula- tion, it appeared perfectly normal and had a weight of 2,400 gms. On this date it was inoculated intracerebrally with fresh passage virus (Zwick strain) and survived, while the control rabbit (Rabbit 68a, weighing 1,150 gms.) inoculated by the same route fell ill on the 20th day following upon infection, and died on the 25th day. Characteristic lesions were found throughout the central nervous system on micro- scopic examination. Rabbit 223a was therefore immune against infection with the strain of equine origin. On the 12.5.27 (i.e. 202 days after the first inocula- tion, and 148 days after the second inoculation with the equine strain, it was inoculated intracerebrally with the strain of ovine origin. At the same time a control rabbit was similarly inoculated. Rabbit 223a continued in health, and gained in weight. The control showed symp- toms of Borna disease 20 days after the inoculation, and died 27 days after the injection. The usual characteristic lesions were found in sections of the central nervous system of this control rabbit, and a portion of the brain infected another rabbit. Experiment 2. The fact that repeated inoculations of fresh virus into the veins of rabbits may immunize them against subsequent intracerebral inoculation has already been referred to (see p. 71). We prepared rabbits by vaccinating in this way with a virus of ovine origin.! A fresh virulent emulsion of brain was centrifugaKzed for 5 minutes. The supernatant fluid was carefully pipetted off and inoculated in the marginal vein of the ear of rabbits, care being taken that the injection was carried out very slowly. Babbit 67. Weight 1,580 gms. 12.5.27. First injection of 2 corns, of a virulent centrifugalized emulsion into the vein. 16.5.27. Weight 1,600 gms. Second injection of 3 corns, of a virulent oentri- fugaUzed emulsion into the vein. 21.5.27. Weight 1,680 gms. Animal normal. 30.5.27. Weight 1,700 gms. Weight normal. 5.6.27. Weight 1,800 gms. Animal normal. 7.6.27. Third injectimi of 5 o.cms. of a virulent centrifugalized emulsion into the vein. ' Several animals were prepared in the same way, but we give below the protocol of the rabbit which served for an experiment of cross immunity between the equine and ovine strain. 76 BORNA DISEASE AND 8.6.27. Weight 1,690 gms. Animal normal. 13.6.27. Weight 1,820 gms. Fourth injection of 3 c.cms. of a virulent centri- fugalized emulsion into the vein. 22.6.27. Weight 1,870 gms. Animal normal. On 23.6.67 the animal, together with a control rabbit, was inoculated intracerebrally with an emulsion of fresh virus of equine origin. Rabbit 67. Weight 1,870 gms. 30.6.27. Animal nonnal. Weight 1,980 gms. 7.7.27 l.l.i,!. 14.7.27. 2,200 20.7.27. 2,150 25.7.27. 2.100 28.7.27. 2,000 15.8.27. 2,100 29.8.27. 2,150 Control. Rabbit 178ii. Weight 1,770 gms. 30.6.27. Normal. Weight 1,820 gms. 7.7.27. „ „ 1,850 „ 14.7.27. „ „ 1,800 „ 18.7.27. Commencement of the disease. Weight 1.650 gms. 20.7.27. Typical svmptoms of the disease. Weight 1,540 gms. 25.7.27. Severely ill. Weight 1,300 gms. 26.7.27. Found dead 33rd day after inoculation. Microscopical examination of sections of the central nervous system — charac- teristic lesions. This experiment shows that the virus of ovine origin immunizes against that of equine origin. Experiment 3. A rabbit (81s) which had resisted inoculation with virulent virus of ovine origin, by scarification of the cornea, was inoculated intracerebrally 110 days later with fresh rabl)it passage virus of equine origin. At the same time a control rabbit ('25a) was inoculated intracerebrally with the same fresh passage virus. Rabbit 81s remained well, while the control, 25a, showed typical symptoms of Borna disease 32 days after inoculation, and died on the 4Sth day. In tliis case also the virus of ovine origin innnunized Rabbit 81s against the pathogenic action of the virus of equine origin introduced into the brain. Conclusion. From the above experiments the following conclusions can be drawn: (1) Rabbits which have become resistant to the virus of enzootic encephalo-myelitis of equine origin prove also to be refractory to infection by intracerebral inoculation of the virus of ovine origin; the converse is also true. (2) Rabbits which have become immunized against enzootic encephalo-myelitis keep this acquired inimunity for at least 148 days. If the immunity is reinforced by repeated inoculations it may last at least 203 days. ENZOOTIC EXCEPHALO-MYELITIS 77 V. Experiments on Cross Immunity between the Virus of Enzootic Encephalo-myelitis and other Viruses of the Filter-passing Group. A. Herpes} On the basis of the following experiment we arrived at the conclu- sion pubhshed as a preliminary note in the British Journal of Experi- mental Pathology (1927) that rabbits immunized against the virus of herpes are still susceptible to infection with the virus of Borna disease. Four large rabbits, 111a, 183a, 185a, and 187a were immunized against herpes. On 7.6.27 their immunity was tested by the intra- cerebral inoculation of fresh herpetic virus; they resisted such infection, while the control rabbit, No. 148a, infected by the same route, died on the 6th day. Sixteen days later the four surviving rabbits were inoculated intracerebrally \vith the virus of encephalo- myehtis, together with a control rabbit. No. 182a. All the rabbits showed symptoms typical of experimental Borna disease and died ; the results are recorded below. Ho. of rabbit. Date of death. CvMure of brain. \ Lesions. lllA 183a 185 A 187 a Control Rabbit 182a Died on the 40th day after inoculation „ 32nd „ 31st „ 32nd ., 40th Negative Typical. Conclusion. No cross immunity exists between herpes and enzootic encephalo-myehtis. B. Rabies.^ On 9.9.27 two rabbits proved to have been immunized against the virus of Borna disease (Babbit 223a, see p. 75, and Rabbit 67, see p. 75), together with, as a control. Babbit 41a, were inoculated intracerebrally with a 'street' virus of rabies. The two rabbits, 223a and 67, which were immune to Borna disease, became paralysed on the 8th day after the inoculation with rabies and died on the 10th day; cultures from the brain were negative. Negri bodies were found in sections of the Ammon's horn of both rabbits. The control rabbit became paralysed after 8 days and died on the 12th day; Negri bodies were found in sections of the Ammon's horn. Conclusion. No cross immunity exists between rabies and enzootic encephalo-myelitis. ' We thank Dr. Perdrau, of the National Institute for Medical Research, for kindly putting a strain of this virus at our disposal. ' We thank Dr. Manouelian of the Pasteur Institute, Paris, for his kindness in putting this strain at our disposal. 78 BORNA DISEASE AND C. Polio-myelitis.^ In the experiments with this virus two strains have been used, (1) a strain of low virulence kindly furnished by Professor Mac- intosh, (2) a very virulent strain kindly provided by Professor Petit. This latter strain, which has been utihzed by us in another series of experiments, killed monkeys by intracerebral inoculation as in- dicated below: Table XIII. Monkey (Macacus rhesus) No. 5. Died the 9th day after inoculation. No. 6. „ 12th " No. 13. „ nth No. 15. „ 11th The lesions produced by this strain of polio-myehtis in the central nervous system of monkeys were very intense, neuronophagia was frequently found in the anterior horns of the spinal cord as well as in the paravertebral ganglia. The virus of polio-myehtis taken from monkeys infected experi- mentally is generally considered as non-pathogenic for the rabbit.- In a first series of experiments we gave repeated intracerebral inoculations of poho-myeUtis to young rabbits; some time afterwards the rabbits so prepared were infected by the same route with the virus of enzootic encephalo-myelitis. Table XIV gives the results of this experiment. Thus the four rabbits having received three successive intracere- bral inoculations with the virus of polio-myehtis and one rabbit which received no such inoculation, proved to be still susceptible to infection with the virus of Borna disease. The conclusion that absolutely no cross immunity exists between the virus of polio-myehtis and that of Borna disease, however, is weakened by the following experiment on a monkey. Monkey M. 2 {Macacus rhesus), which had apparently almost com- pletely recovered from an intracerebral inoculation of tlie virus of Borna disease, after having shown the morbid symptoms recorded on p. 36, was inoculated into the brain with a passage virus of polio- myehtis (strain Petit). At the same time two controls of a comparable size and the same species were similarly inoculated. The subsequent history of the two controls was as follows : M. 13 {Macacus rhesus) 5.10.27. Inoculated intracerobrally with the viru.s of polio- mj-elitis. 10.10.27. Normal. 11.10.27. Paralysed, found procumbent. 12.10.27. ' Our thankn are due to M. le Prof. A. Petit, of the I'iMit<olio-niyeliti« is not (jathogcnic for rahbitii. ENZOOTIC ENCEPHALO-MYELITIS 79 "5 . "3 . u 11 It .2-3 •^ "3 "eS •«' 3 T- 1^ a. 2 IS 'S. 1 'S. >> 1 iz 7" o '^ ■*^ .So .SaJ aj oj CO oj s.s |.s "3 2 fe^i !3 g; •z ^ 4 !S d . c >>— d d 05 -•3 41 -a g ^1 fl J- .2 j=.2 c: . c ^ 'aj s % C3 X e5 >, -s> :o q^ 2 ''^ o <0 O Oi (N d c^ — M J lMh-3 aj-a i»T3 2-T3 1:3 E >. E >. S >. E >> s >> o c C :3 D =S o s -»^ M *^ TJ *^ -d -*^ "5 *> T3 P.CC o-« °-^ p^-2 ^ i o. '•^ 0< - a o S 4J S^ s -^ S "5 6:2 «5 >.JS ^'•S ?ic3 >>s >>» >.a5 X M X re X C^ X iq rn'O "S "O x? X "P -s-^ X T3 x"^ t. .Si S5 S3 E5 £3 2 41 £5 ■s i Ca o o 3 o o o 3 n « o tN 05 8 ^ ^ CO ia_ ^ ^. « CO o § 1=5 •2* ~ -^ '^* '^ '- ci IN 05 ^ g- ~T •2 -^ g o O o o lO •o 00 t' 6.9. inoc- irus ■*g « w 1 1 1 ^ ^ ^ ^ " -^ * ^ =^ s-S •2 5 . a e ^ ^ 11 .8 o o o o t^ lO o o 3l^ :« Ca o M 1 1 1 •S* ■^ 3 S £ ^ S ^ -rf£ S '<• S 5 g ^* '-2 "s ^- O"^ o s §1 ■i 8 g g s 1 c o ; »» •» « ^ CO S> CD I> .&> Q 5| ^ *o*.*-* < ■< -< < < • -S ■^ 1--5 o o> o -o o O o N CD CO ^g 80 BORXA DISEASE AND 13.10.27. Paralysed, found procumbent. 14.10.27. 15.10.27. 16.10.27. Found dead, 11th daj-s after inoculation. Cultures of brain. Negative. Sections. Lesions typical of polio-myelitis. M. 15 {Macacos rhesus) 5.10.27. Inoculated intracerebrally with the virus of polio-myelitis. 10.10.27. Normal. 11.10.27. 12.10.27. 13.10.27. 14.10.27. Paralysis of the left arm. 15.10.27. Found procumbent and paralysed. 16.10.27. Found dead 11th day. Cultures of brain. Negative. Sections. Lesions typical of polio-myelitis. Monkey M. 2 {Macacos rhesus), which had survived the inoculation with Borna disease, when inoculated with polio-myelitis, behaved as follows : 5.10.27. Inoculated into the brain with the virus of polio-myelitis 130 days after the inoculation with the virus of Borna disease. 10.10.27. No added symptoms up to this date. 15.10.27. 17.10.27. 19.10.27. 20.10.27. Fifteenth day, myoclonic movements. 21.10.27. Epileptiform trembling movements. 22.10.27. Slight paresis of the hind quarters. 23.10.27. Paresis more marked. 24.10.27. Total paralysis of the hind quarters and partial paralysis of the fore- limbs. 25.10.27. Could make movements with the head, but the total paralysis of the arms and legs prevented it from getting up. 26.10.27. Same condition. 27.10.27. Same condition, but still feeding. 28.10.27. 29.10.27. Animal in a comatose state. 30.10.27. 31.10.27. Died 26 days after the inoculation with the virus of polio-myelitis and 156 days after the infection with Borna. We have drawn attention in the course of this monograph to the rosomblance between the symptomatology and histological picture in polio-myelitis in the monkey and experimental enzootic encephalo- myelitis in th(! same species. The dilferences i)etween tiieni are the longer period of incubation and the slower evolution and less in- tensity of the lesions in the nervous system in the latter dis(>ase. Monkey M. 2 died after a lapse of time nuich longer than the averag(! for animals inoculated with polio-myelitis, the strain used generally killing the species of animal in from !) to 11 days. We had, therefore, to determiiH? whether it died from polio-myelitis or from a persisting infect ifni of I'orna disease. On an anatomo-puthological ENZOOTIC ENCEPHALO-MYELITIS 81 study of the various parts of the nervous system of this monkey the lesions of experimental enzootic encephalo-myelitis were found, includ- ing the corpuscles of Joest-Degen. The general aspect of the altera- tions in the lumbar region of the spinal cord indicated that these were produced by the virus of Borna disease and not by the virus of polio- myelitis. This conclusion received further support when emulsions from various parts of the nervous system were passaged to fresh animals. The results of these inoculations were as follows: (1) Eabbit 460a, weighing 2,320 gms., and Rabbit 462a, weighing 1,740 gms., were inoculated intracerebrally with an emulsion of the brain of Monkey M. 2. The former died on the 37th day and the latter on the 38th day after infection, and lesions characteristic of experi- mental Borna disease were found in sections of the various parts of the nervous system of both animals. A passage was made with the brain of Rabbit 462a to a fresh Rabbit 405a, weighing 1,330 gms. Eabbit 40.5a succumbed on the 42nd day of Borna disease. (2) Rabbit 464a, weighing 1,960 gms.. Rabbit 458a, weighing 1,760 gms., and Rabbit 459a weighing 2,500 gms., were inoculated intracerebrally with an emulsion of the cervical region of the spinal cord of Monkey M. 2. Rabbit 464a died on the 34th day, 458a on the 30th day, and 459a on the 30th day after inoculation, and lesions typical of Borna disease were found in sections of the nervous system of all three. A passage was made with the brain of Rabbit 458a to a fresh rabbit, 414a, which died on the 33rd day of experimental enzootic encephalo-myelitis. (3) Eabbit 454a, weighing 1,960 gms.. Rabbit 455a, weighing 1,960 gms., and Rabbit 463a, weighing 2,300 gms., were inoculated intracerebrally with an emulsion of the lumbar cord of Monkey M. 2. The first died on the 40th day, the second on the 35th day, and the third on the 34th day of experimental Borna disease. Rabbit 415a, weighing 1,880 gms., when inoculated intracerebrally with an emul- sion of the brain of Rabbit 463a, died of Borna disease on the 30th day. The results of these rabbit inoculations proved that the virus of Boma disease still existed in the brain and spinal cord of Monkey M. 2. Monkey M. 20 {Macacus rhesus) was inoculated with an emulsion of a portion of the brain and the cervical cord of Monkey M. 2. It remained healthy for a period of 60 days, when the commencement of paresis of the hind quarters was first observed. Paresis later became accentuated and reached the fore hmbs. The animal died on the 68th day after inoculation, and the lesions of experimental enzootic ence- phalo-myehtis were found in sections of the central nervous system. Rabbits 340a and 341a were inoculated intracerebrally with an emul- sion of the brain of Monkey M. 20, and both died of experimental Boma disease. In resume, the activity of virus of Borna disease which had become dormant in the central nervous system of Monkey M. 21 , 130 days after the inoculation, as indicated by the clinical picture, was revived by the introduction of the virus of polio-myelitis by the intracerebral route. As a result of the second infection the monkey died and we 6409 „ 82 BORNA DISEASE AND demonstrated the presence of the virus of Borna disease in its brain and spinal cord, but the virus of poho-myehtis had been destroyed. Without wishing to attach too much importance to this single experiment, we are forced to conclude that the monkey which sur- vived for 130 days the infection with Borna disease showed an exceptional resistance of an anomalous character to the virus of polio-myehtis. This resistance might be the result of a certain degree of immmiity conferred by the first infection. On the other hand, it might be explauied in another way. The experiments by Gilde- meister and Herzberg (1925) indicate that guinea-pigs which showed lesions on the pads of the metatarsus as a result of infection with the virus of vaccinia develop a certain local resistance to infection with herpetic virus inoculated by the same route. The authors con- clude that there is a certain degree of immunity conferred by one virus against infection with the other. In a similar way, certain rabbits, which after recovering from vaccinal keratitis, when inocu- lated some time later on the same cornea with the virus of herpetic encephaUtis, sometimes resist the second infection. We do not consider in these circumstances that an inununity results in the proper sense of the word, but rather a local mobiUzation of the elements of defence provoked by the first virus inoculated, thus conferring on the tissue a certain degree of resistance of a non- specific character which does not exist normally. We intend to repeat our experiment. In addition, monkeys are being immunized against polio-myehtis which will be inoculated later with the virus of Borna disease. VI. SlMMARV. A solid immunity can occasionally be obtained against Borna disease in the rabbit by inoculating suitably attenuated virus into the brain. Multiple intravenous injections, infection by corneal scarification, or intratesticular inoculation with fresh virus, can also produce im- munity. We have not succeeded in producing inununity by inoculating virus killed by chloroform, ether, or ultra-violet light intracerebrally into rabbits. Multiple inoculations subcutaneously of large quantities of for- molized virus leads to immunity in a Umited number of animals. Rabbits immunized against an equine strain of the virus of Borna disease were resistant to intracerebral infection with an ovine strain and vice versa. No cross immunity was obtained between Borna disease and herpes or rabies. Cross inununity between Borna disease and pt)lio-niyeliti9 was not observed when rabbits were the subject of experiment, but in an experiment carried out on a monkey the result suggested that some resistance to the virus of polio-myelitis may be produced by a previous attack of experimental Borna disease. ENZOOTIC ENCEPHALO-MYELITIS 83 13. CHEMOTHERAPY Various medicaments, calomel, mercuric chloride, salvarsan, atoxyl, have been tried in the treatment of enzootic encephalo- myelitis. The greatest success as regards the treatment by drugs has been claimed for the administration of urotropine (hexamethy- lenetetramine). Moussu and Marchand (1926) obtained remarkably good results in the treatment of the disease in horses and cattle by inoculation of urotropine intravenously. According to these authors, when 15 to 20 gms. is administered on the appearance of the first symptoms, the mortahty in epizootics may be lowered from 80 or 90 per cent, to 25 per cent. In Germany Ostertag (1924) has generahzed the use of urotropine on a large scale in the treatment of enzootic encephalo-myehtis. H. Bohn (1927) inoculated 30 gms. per day for 4 to 5 days intravenously into affected horses with good results. Trepel (1926) advocates the use of a total of 100 gms. during the course of a few days. Grimm (1927) obtained a smaller number of recoveries than the authors cited above. All authors appear to be agreed as to the beneficial action of the drug in Borna disease of the horse. We have carried out experiments to see whether urotropine was as efficacious in treating the disease in the rabbit as in the horse. We commenced by determining the maximum intravenous dose supported by a rabbit. This appeared to be about 0-5 gms. per kgm., a dose which can be repeated at least eight times at 4 to 5 days' interval. Subsequently the experiment recorded in Table XV was carried out. Summary. The dose of urotropine we gave to the rabbit was equivalent, weight for weight, to 100-200 gms. for the horse and to a total amount of 600 gms., yet we were not able to demonstrate any prophylactic or curative action. P 2 84 BOENA DISEASE AND Result. 5" i i .1 >> ^>. 1* ll l" "> U 3 X 24.7.27. Typical symptoms. Weight 1,550 gms. Typical disease. Weight 1,000 gms. Typi(!al disease. Weight 1,050 gms. 1 11 1 1 in. 7. 27. Urotropine tnoca- laled into veins. Animal showing symptoms. Weight 1,880 gms. Weight 1,900 gms. Typical disease. Urotropine O-S gr. Typii^al (tomraence- ment of disease. Weight 2,100 gms. Urotropine 0-8 gr. Typical symptoms \Veight l^fiOOgms. No urotropine i- ■s 2 U.7.27. Urotropine inocu- lated into veins. Weight 1,900 gms. Urotropine 0-8 gr. Weight 2,000 gm's. Urotropine 0-8 gr. Weight 2,200 gms. Urotropine 0'8 gr. Weight 1,800 gms. No urotropine 1 .2? e^ Mx c ° u Weight 2,0.50 gms. Urotropine 0-8 gr. Weight 2,300 gms. Urotropine 0-8 gr. Weight 1,850 gms. No urotropine Weight 2,,320 gms. Urotropine 1 gr. 2.7.27. Urotropine inocu- lated into veins. Weight 1,950 gms. Urotropine 0-8 gr. Weight 2,100 gms. Urotropine 0-8 gr. Weight 2,250 gms. Urotropine 0-8 gr. E ^§ II U 28.6.27. Urotropine inocu- lated into veins. Weight 2,020 gms. Urotropine 0-8 gr. Weight 2,150 gms. Urotropine 0-8 gr. Weight 2.200 gms. Urotropine 0-8 gr. Weight 1,820 gms. No urotropine a&) 3i U Inoculation of virus intracere- brally. 23.6.27. Urotropine inocu- lated into veins. §4 a-x «.| •a 5. ^ £ ■n Weight 2.100 gms. Urotropine 0-8 gr. Weight 2,220 gms. Urotropine 0-8 gr. Virus into brain. Weight 1,770 gms. No urotropine Not inoculated with vims. Weight 2,400 gms. Urotropine i gr. 1 18U 179a Control 178a 1< ENZOOTIC ENCEPHALO-MYELITIS 85 REFERENCES Alberttn, a. (1926). — Thesis. Lyons. Alvisatos, G. p. (1922). Deutsche med. Wchnschr., 48, 295. Baudoin, a., and Schaeffer, H. (1926). Paris Med.. 16, 249. Beck, A. (1925). Ztschr.f. Infektioiisk. d. Hausliere, 28, 99. Beck, A., and Frohbose (1926). Archiv f. wissensch. u. prald, Tierh., 54, 84. B^RIEL, L. (1926).— i(/o7i med., 1, 667. BtRiEL, L., and Devic, A. (1926). Li/on med., 1, 671. BoHN, H. (1927). Deutsche tierarztl. Wchnschr., 35, 584. Causel, M. (1924). Eec. med. vet., 100, 526. Christiani (1909). Arch. f. ivissensch. v. prakt. Tierh., 35, 253. Dahm (1909). Miiiichen. med. Wchnschr., 56, 2553. David and Dekester (1926). Bull, et mem. Soc. med. d'hop. de Paris, 3, S., 50, 75. Dexler, H. (1900). Ztschr.f. Thiermed., 4, 110. DiOBY, K. (1919). Immunity in Health. 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Bert, tierdrzll. Wchnschr., 41, 129. ZwiCK, W., Seifried, 0., and Witte, J. (1926). Ztschr.f. Inftklionskr. d. Uaustiere, 30,42. DESCRIPTION OF PHOTOGRAPHS AND COLOURED PLATES Fig. 1. Photograph of Rabbit No. 275. 29 days after intracerebral infection with the virus of Borna disease, and 3 days before death. The characteristic position of the head and ears is depicted. Fig. 2. Photograph of 7l/aono?(« rAes«.« No. 1. 04 days after inoculation. Paralysis of the hind quarters; paresis of the right arm, the hand not being able to grip any object presented to it. Fig. 3. Photograph of Macaciis rhesus No. 1. 69 days after inoculation. Charac- teristic ' hunched up ' attitude. Fig. 4. Photograph of Ma(acus rhesus No. 2. .51 days after the infection by the intra- cerebral route. Facial paralysis on the left side. Fig. 5. Photograph of Macacus rhesus No. 2. 67 days after inoculation. 3rd crisis of the disease. Facial paralysis on the right side. Fig. 6. Photograph of Guinea-pig 9.5k. 60 days after intracerebral inoculation, and 5 days before death. Paralysis of the hind quarters. Fig. 7. Photograph of Guinea-pig 1a. 50 days after intracerebral inoculation, and 2 days before death. General paralysis. Fig. 8. Photograph of Rat No. 14. 63 days after inoculation intraeerebrally, and 4 days before the death of the animal. At this stage the rat placed on the flank made several useless efforts with the fore-legs to recover its normal position. Fig. 9. Photograph of the stomach wall of Rabbit 212 which died 44 days after the intracerebral inoculation, showing the haemorrhagic lenticular areas. L = greater curvature of the stomach; P = lenticular haemorrhagic areas. Fig. 10. Microphotograph, x 160. Rabbit 209 dead 71 days after intratesticular inoculation. Meningitis with mononuclear cells with ' cuffing ' around the vessels. V = lumen of blood-vessel; C = cerebral cortex; M = mononuclear leucocj'te of inflam- matory process. Fig. 11. Microphotograph, x 300. Rabbit 51a dead 28 days after intracerebral infection. Perivascular 'cuff' in the cerebral cortex cut longitudinally. V = lumen of blood-vessel ; E = endothelium of blood-vessel ; M = mononuclear leucocytes taking part in the infiltrative process. Fig. 12. Microphotograph, -^165. Rabbit 11d dead 27 days after intracerebral inoculation. Section showing the aspect of the Cornu Amynoyiis. F = area of infiltrating lymphocytes between the nerve-cells; L = infiltrating lymphocytes; C = cells containing the corpuscles of Joest-Degen within their nuclei. Fig. 13. Microphotograph, X 800. Rabbit 25 dead 35 days after cerebral infection. 'Cyst' in a nerve-cell of the Ammon's horn. C = 'cyst'; N = nucleus; n=nucleolus. Fig. 14. Microphotograph, x 1,000. Rabbit 80a dead 28 days after inoculation intraeerebrally. ' Cyst " in the protoplasm of a nerve-cell of the Ammon's horn. C = 'cyst'; N = nucleolus. Fig. 15. Microphotograph, x 900. Rabbit 1 Id dead 27 days after cerebral infection. 'Cyst' in the protoplasm of a nerve-cell of the Cornu Amman is. Fig. 16. Microphotograph, x 1,000. Rabbit 51a dead 28 days after inoculation into the brain. 'Cyst' in the protoplasm of a nerve-cell in the Cornu Arnmonis. Fig. 17. Microphotograph, x 1,000. Rabbit No. 22 dead 24 days after cerebral infection. Nerve-cell in medulla oblongata showing neuronophagia. C = degenerated cytoplasm ; p = polj'morphonuclear penetrating neuron ; m = mononuclear cells. Fig. 18. Microphotograph, x 230 (enlarged two diameters). Rabbit 295 dead 19 days after intracerebral inoculation. Section in the region of the pons showing (d) degener- ated nerve-cells, (s) ' salellitism' with commencing neuronophagia, (n) neuronophagia, (1) lymphocytes in the parenchyma. 88 DESCEIPTIOX OF PHOTOGRAPHS, ETC. Fig. 19. Microphotograph, x 1,000. Rabbit 278 inoculated iutracerebrally and sacrificed 20 days later. Commencing neuronophagia in the anterior horn of the spinal cord in the lumbar region. N = nucleus of nerve-cell: P = cytoplasm; M = mononuclear cells. Fig. 20. Microphotograph, x 66. Rabbit 44 dead 33 days after intracerebral inocula- tion. Section of the lumbar region of the spinal cord. .S = cord; M = perivascular "cuff' in a nerve-trunk; I = interstitial mononuclear infiltration; A = accumulation of mono- nuclear leucocytes. Fio. 21. Microphotograph, x 66. Rabbit No. 30 dead 36 days after the inoculation given intracerebrally. Spinal ganglion in the lumbar region. N = ganglion nerve-oell in a state of neuronophagia; I =^ formation of a nodule of mononuclear leucocytes which has formed at the site where a nerve-cell is undergoing neuronophagia; V = perivascular infiltration. Fio. 22. Microphotograph, x 66. Rabbit 50a dead 53 days after cerebral inocula- tion. Section of the cord in the lumbar region including a spinal ganglion. V = vacuolization in certain ganglion cells; I = interstitial infiltration; N — neuronophagia ; P = perivascular infiltration. Fio. 23. Microphotograph, x 1,000. Rabbit 164 dead 29 days after intracerebral inoculation. Spinal ganglion in the lumbar region. Neuronophagia with the formation of nodules consisting of mononuclear cells. R = remains of a destroyed neuron; V = vacuole in a nerve-cell; P = polymorphonuclear; L = lymphocytes. Fig. 24. Microphotograph, x 950. Rabbit No. 30 dead 36 days after intracerebral inoculation. Spinal ganglion in the lumbar region. Degeneration of a ganglion cell and commencing neuronophagia. C — degenerated cell which has become o.xyphilic and is on the way to destruction; P- polymorphonuclear cells; L'^ lymphocytes; M = mono- nuclears participating in the process of neuronophagia. Fio. 25. Microphotograph, x 1,000. Rabbit 164 dead 29 days after intracerebral inoculation. Spinal ganglion in the thoracic region. N-^ neuronophagia; G — particle of chromatin probably originating from a pyknosed polymorphonuclear; M -macro- phage; C = cell — commencing neuronophagia; L lymphocytes. Fio. 26. Microphotograph, x 8(M). Rabbit No. 30 dead 36 days after cerebral in. oculation. Spinal ganglion in the lumbar region. M == mononuclear cells infiltrating; D = commencing neuronophagia; V - vessel surrounded by perivascular 'cuff'. Fio. 27. Microphotograph, :■ 5. Rabbit 140a dead 49 days after intracerebral infec- tion with glycerinated virus of passage. Section of the terminal part of the spinal cord with roots of the sciatic nerve and corresponding ganglion. M =spinal cord; R nerve- root; G ^spinal ganglion; .S" sciatic nerve. In sections cut in this way the lesions in the regions mentioned above can be seen as a whole. Fio. 28. Microphotograph, v 350. Rabbit 140a. Longitudinal section of the sciatic nerve near the popliteal region. A perivascular 'cuff' is seen. V lumen of vessel. M = infiltrating mononuclear cells; L lymphocytes in the thickness of the nerve. Fio. 29. Microphotograph, x l.iO. MararunrhenunNoA. Sectiim of the parietal lobe; C = cerebral corte.i; M ^ mononuclear meningitis; V blood. vessel cut longitudinally with walls infiltrated with mononuclear cells. Fio. 30. Microphotograph, :• 120. Mararu-i rhesng No. 3. Periva.scular «el in the centre (very intense process of perivascular infiltration). V l)lood.ves.sel; K nodule formed by monimudear cells. Kio. 32. Microphotograph, ^360. Maranis rhesun No. I. Section through basal ganglia. Small area of monimuclear cells. N neuron; L lymphocytes. Fio. 33. Microphotograph, > 150. MiirncuA rhemn'iin.X. Vascular infiltration in the terminal part of the spinal cord. V lumen of blocnl. vessel; M muscular coat; I' infiltrating mononuclear cells. Fio. 34. Microphotograph, > 3tMJ. Macacun rhtDU-i tio. \. Posterior root of sciatic nerve. Interstitial infiltrati(m and perivasc'ular 'i^ufliiig'. V lumen of bloud-vosaci; M mononuclear cell in the |)rocess of |>eriviiscular 'cutting'; in macrophage. Fig. 35. Microphotograph, • "(H). Marncun rheaux No. 3. Spinal ganglion in cervical region. Corpuscles (type Joest-Degen) surrtiunded by a halo in the nerve-cells of the ganglion. C intranuclear corpuscle of .loest.Degen; N nucleolus. DESCEIPTION OF PHOTOGRAPHS, ETC. 89 Fig. 36. Microphotograph, ( ;< 3(5, enlarged 2t diameters). Maearus rhesus No. 1. Section of spinal ganglion in lumbar region of spinal cord showing the profound changes which predominate the peripheral parts of the nervous system. (i) = intranuclear cor- puscle (Joest-Degen) with surrounding halo; n = nodule of mononuclear cells replacing destroyed neuron; d = degenerated ganglion cells; np = neuronophagia ; ic = peri- cellular infiltration; it = interstitial infiltration; c = capsule of ganglion. Fig. 37. Microphotograph, x 55. ilacacus rhesus No. 3. Section through the brachial nerve. Perivascular 'cuffing' and interstitial infiltration. G = nerve sheath; V = perivascular 'cuffs'; I = interstitial infiltration. Fig. 38. Microphotograph, x460. Macacus rhesiisNo. \. Transverse section of the sciatic nerve several centimetres from its exit from the greater sciatic foramen — peri- vascular 'cuff'. V = lumen of blood-vessel; E = vascul^ endothelium; L = lympho- cytes. Fig. 39. Microphotograph, x55, (photograph enlarged 2A diameters). Macacus rhesus No. 1. Transverse section of sciatic nerve after its exit from the greater sciatic foramen. V = large vessel with perivascular infiltration ; v = small vessels showing same phenomenon; 1 = Ij'mphocytes (interstitial infiltration). COLOURED PLATES PLATE I Fig. 1. Staining, toluidin blue-eosin. Obj. 5 mm., oe. 2, x260. Rabbit 295 dead 19 days after intracerebral infection. CelliJar degeneration in the medulla oblongata. N = neuron in normal state; C = degenerated nerve-cell — ■ tigrolysis-oxyphilia — nucleus eccentric; E = degenerated neuron; F = fragment of nerve-cell; L = lymphocytes. Fig. 2. Staining, Mann. 1 12 oil immersion, oc. i. Rabbit 25 dead 35 days after cerebral inoculation. Oxyphilic corpuscle surrounded by a halo in a nerve-cell in the Ammon's horn. C = corpuscle (type Joest-Degen) sur- rounded by a halo. Fig. 3. Staining, Mann. 1/12 oil immersion, oc. 4. Rabbit 80.i dead 28 days after the inoculation into the brain. N = nucleus of neuron repulsed by the 'cyst'; n = nucleolus; C = 'C'yst\ this 'cyst' is the degenerated nucleus of a mononuclear cell which has succeeded in penetrating the nerve-cell. Fig. 4. Staining, toluidin blue-eosin. 1/12 oil immersion, oc. 4. Guinea-pig 85e dead 82 days after cerebral infection. Section of medulla oblongata. N = nucleolus; C = corpuscle of Joest-Degen; H = halo around the corpuscle. Fig. 5. Staining, Mann. 1 12 oil immersion, oe. 4. Rabbit 209 dead 71 days after the inoculation of the virus into the testicle. Neuron of the anterior horn of the spinal cord in the lumbar region. C = Intranuclear cor- puscle (.Joest-Degen): N=nucleolus. PLATE II Fig. 1. Staining, toluidin blue-eosin. 1/12 oil immersion, oc. 1. Rabbit 243 dead 22 days after inoculation into the brain. Section of the cord anterior horn shoiving degeneration of the nerve-cells. V = small blood-vessel; P = protoplasm of degenerated nerve-cells; D = protoplasm debris; 0 = vacuole in the cytoplasm of a degenerated neuron ; C = cytoplasm ; L = lymphocytes. Fio. 2. Staining, Mann. 1/12 oil immersion, oc. 4. Macacus rhesus No. 1. Neurons of the anterior horn of the thoracic region of the cord containing corpuscles (.Joest-Degen type). N = nucleus of the nerve-cell; n = nucleolus; C' = corpuscle (Joest-Degen) surrounded by a halo. At the periphery of the corpuscle a blue staining area is seen. PLATE III Fig. 1. Staining, Mann. 1/12 oil immersion, oc. 2. Macacus rhesus No. 1. PyTamidal cells of the frontal lobe containing oxyphilic cor- puscles surrounded by a halo. N = nucleolus; C = corpuscle (Joest-Degen). Fig. 2. Staining, Mann. 1/12 oil immersion, oc. 4. Rabbit 298 dead 39 days after intracerebral inoculation. Cervical region of the cord : anterior horn. Formation of intranuclear corpuscles in the interior of the nucleus of the neuron. N = nucleolus; C' = oxyphilic corpuscles; a study of the process of the 90 DESCRIPTION OF PHOTOGEAPHS, ETC. formation of the corpuscles of Joest-Degen has shown that several small oxyphilic corpuscles fuse together to form one large corpuscle stirrounded by a halo. Fig. 3. Staining, orange G. eosin, polychrome blue (Unna). 1 12 oU immersion, oc. 4. Guinea-pig 99k dead 19 days after intracerebral inoculation. A lymphocj-te has penetrated into the cytoplasm of a nerve-cell of the Cornu Amnionic. N = nucleolu8; L = lymphocytes. Fig. 4. Staining, Mann. J 1 12 oil immersion, oc. 4. 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